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Sample records for residual circulation cells

  1. Early recovery of circulating immature B cells in B-lymphoblastic leukemia patients after CD19 targeted CAR T cell therapy: A pitfall for minimal residual disease detection.

    Science.gov (United States)

    Xiao, Wenbin; Salem, Dalia; McCoy, Catharine S; Lee, Daniel; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M

    2017-09-09

    CD19-targeted chimeric-antigen receptor-modified T-cells (CAR-T) are promising in the treatment of refractory B-lymphoblastic leukemia (B-ALL). Minimal residual disease (MRD) detection by multicolor flow cytometry (FCM) is critical to distinguish B-ALL MRD from regenerating, non-neoplastic B-cell populations. FCM was performed on samples from 9 patients with B-ALL treated with CAR-T. All 9 patients showed response to CAR-T. Additionally, FCM revealed circulating CD10 + B cells, potentially mimicking MRD. Circulating CD10+ B-cells were detected in blood from 3 days to 3 months after CAR-T, comprising 73% (median) of B-cells (52-83%, 95%CI). They expressed CD19, CD10, CD20, bright CD9, CD22, CD24, moderate CD38 and dim CD58, but were CD34 (-), with bright CD45 and polyclonal surface light chain immunoglobulin (sIg) expression. A similar CD10 + B-cell subpopulation was detected by marrow FCM, amidst abundant B-cell precursors. These circulating CD10 + B-cells are compatible with immature B-cells, and are a reflection of B-cell recovery within the marrow. They are immunophenotypically distinguishable from residual B-ALL. Expression of light chain sIg and key surface antigens characterizing regenerating B-cell precursors can distinguish immature B-cells from B-ALL MRD and prevent misdiagnosis. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  2. Circulating microRNA levels predict residual beta cell function and glycaemic control in children with type 1 diabetes mellitus

    DEFF Research Database (Denmark)

    Samandari, Nasim; Mirza, Aashiq H.; Nielsen, Lotte B.

    2017-01-01

    from the Danish Remission Phase Cohort, and profiled for miRNAs. At the same time points, meal-stimulated C-peptide and HbA1c levels were measured and insulin-dose adjusted HbA1c (IDAA1c) calculated. miRNAs that at 3 months after diagnosis predicted residual beta cell function and glycaemic control...... in this subgroup were further validated in the remaining cohort (n = 83). Statistical analysis of miRNA prediction for disease progression was performed by multiple linear regression analysis adjusted for age and sex. Results: In the discovery analysis, six miRNAs (hsa-miR-24-3p, hsa-miR-146a-5p, hsa-miR-194-5p...

  3. Unmasking circulating tumor cells

    NARCIS (Netherlands)

    Swennenhuis, Joost Franciscus

    2017-01-01

    The number of Circulating Tumor Cells (CTCs) that can be isolated from blood of cancer patients is prognostic for the course of the disease. A higher number of CTCs correlates with a worse prognosis. A change from a higher number to a lower number of CTCs indicates a benefit of the current treatment

  4. Enhanced residual mean circulation during the evolution of split type ...

    Indian Academy of Sciences (India)

    8

    keywords: split events, stratospheric sudden warming, residual mean circulation. 1 Introduction ... sudden warming. It is characterized by a rapid cooling of the polar cap tempera- ture (Kuroda, 2008). The competition between planetary waves and gravity waves to the residual .... any automated scheme. The split events ...

  5. Residual estuarine circulation in the Mandovi, a monsoonal estuary: A three-dimensional model study

    Digital Repository Service at National Institute of Oceanography (India)

    Vijith, V.; Shetye, S.R.; Baetens, K.; Luyten, P.; Michael, G.S.

    then analyse the model results to define characteristics of residual estuarine circulation in the Mandovi. Our motivation to study this aspect of the Mandovi`s dynamics is derived from the following three considerations. First, residual circulation is important...

  6. Circulating Levels of MicroRNA from Children with Newly Diagnosed Type 1 Diabetes and Healthy Controls: Evidence That miR-25 Associates to Residual Beta-Cell Function and Glycaemic Control during Disease Progression

    Directory of Open Access Journals (Sweden)

    Lotte B. Nielsen

    2012-01-01

    Full Text Available This study aims to identify key miRNAs in circulation, which predict ongoing beta-cell destruction and regeneration in children with newly diagnosed Type 1 Diabetes (T1D. We compared expression level of sera miRNAs from new onset T1D children and age-matched healthy controls and related the miRNAs expression levels to beta-cell function and glycaemic control. Global miRNA sequencing analyses were performed on sera pools from two T1D cohorts (n = 275 and 129, resp. and one control group (n = 151. We identified twelve upregulated human miRNAs in T1D patients (miR-152, miR-30a-5p, miR-181a, miR-24, miR-148a, miR-210, miR-27a, miR-29a, miR-26a, miR-27b, miR-25, miR-200a; several of these miRNAs were linked to apoptosis and beta-cell networks. Furthermore, we identified miR-25 as negatively associated with residual beta-cell function (est.: −0.12, P = 0.0037, and positively associated with glycaemic control (HbA1c (est.: 0.11, P = 0.0035 3 months after onset. In conclusion this study demonstrates that miR-25 might be a “tissue-specific” miRNA for glycaemic control 3 months after diagnosis in new onset T1D children and therefore supports the role of circulating miRNAs as predictive biomarkers for tissue physiopathology and potential intervention targets.

  7. Circulating Levels of MicroRNA from Children with Newly Diagnosed Type 1 Diabetes and Healthy Controls: Evidence That miR-25 Associates to Residual Beta-Cell Function and Glycaemic Control during Disease Progression

    DEFF Research Database (Denmark)

    Nielsen, Lotte B.; Wang, Cheng; Sorensen, Kaspar

    2012-01-01

    This study aims to identify key miRNAs in circulation, which predict ongoing beta-cell destruction and regeneration in children with newly diagnosed Type 1 Diabetes (T1D). We compared expression level of sera miRNAs from new onset T1D children and age-matched healthy controls and related the mi...... for tissue physiopathology and potential intervention targets....

  8. The influence of the “tidal stress” on the residual circulation

    OpenAIRE

    Nihoul, Jacques C. J.; Ronday, Francois C.

    2011-01-01

    The residual current field in the sea is defined as the mean velocity field over a time sufficiently long to cancel transitory wind currents and tidal oscillations. The hydrodynamic equations governing the residual circulation are established and it is shown that, in the regions of intensive tides, the tidal motion has a cogent influence on the residual flow pattern. This effect which arises from the non linear terms is equivalent to the application of a “tidal stress” which combines with the...

  9. Anelastic Models of Fully-Convective Stars: Differential Rotation, Meridional Circulation and Residual Entropy

    Science.gov (United States)

    Sainsbury-Martinez, Felix; Browning, Matthew; Miesch, Mark; Featherstone, Nicholas A.

    2018-01-01

    Low-Mass stars are typically fully convective, and as such their dynamics may differ significantly from sun-like stars. Here we present a series of 3D anelastic HD and MHD simulations of fully convective stars, designed to investigate how the meridional circulation, the differential rotation, and residual entropy are affected by both varying stellar parameters, such as the luminosity or the rotation rate, and by the presence of a magnetic field. We also investigate, more specifically, a theoretical model in which isorotation contours and residual entropy (σ‧ = σ ‑ σ(r)) are intrinsically linked via the thermal wind equation (as proposed in the Solar context by Balbus in 2009). We have selected our simulation parameters in such as way as to span the transition between Solar-like differential rotation (fast equator + slow poles) and ‘anti-Solar’ differential rotation (slow equator + fast poles), as characterised by the convective Rossby number and △Ω. We illustrate the transition from single-celled to multi-celled MC profiles, and from positive to negative latitudinal entropy gradients. We show that an extrapolation involving both TWB and the σ‧/Ω link provides a reasonable estimate for the interior profile of our fully convective stars. Finally, we also present a selection of MHD simulations which exhibit an almost unsuppressed differential rotation profile, with energy balances remaining dominated by kinetic components.

  10. Circulating immune cells in multiple sclerosis.

    Science.gov (United States)

    Jones, A P; Kermode, A G; Lucas, R M; Carroll, W M; Nolan, D; Hart, P H

    2017-02-01

    Circulating T and B lymphocytes contribute to the pathogenesis of the neuroinflammatory autoimmune disease, multiple sclerosis (MS). Further progress in the development of MS treatments is dependent upon a greater understanding of the immunological disturbances that underlie the disease. Analyses of circulating immune cells by flow cytometry have revealed MS-associated alterations in the composition and function of T and B cell subsets, including temporal changes associated with disease activity. Disturbances in circulating immune populations reflect those observed in the central nervous system and include skewing towards proinflammatory CD4 + and CD8 + T cells and B cells, greater proportions of follicular T helper cells and functional defects in the corresponding T and B regulatory subsets. Utilizing the analytical power of modern flow cytometers, researchers are now well positioned to monitor immunological changes associated with disease activity or intervention, describe immunological signatures with predictive value and identify targets for therapeutic drug development. This review discusses the contribution of various T and B lymphocyte subsets to MS pathogenesis, provides current and relevant phenotypical descriptions to assist in experimental design and highlights areas of future research. © 2016 British Society for Immunology.

  11. Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility?

    Science.gov (United States)

    Gold, Bert; Cankovic, Milena; Furtado, Larissa V.; Meier, Frederick; Gocke, Christopher D.

    2016-01-01

    Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided. PMID:25908243

  12. Circulating Tumor Cells Measurements in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Franck Chiappini

    2012-01-01

    Full Text Available Liver cancer is the fifth most common cancer in men and the seventh in women. During the past 20 years, the incidence of HCC has tripled while the 5-year survival rate has remained below 12%. The presence of circulating tumor cells (CTC reflects the aggressiveness nature of a tumor. Many attempts have been made to develop assays that reliably detect and enumerate the CTC during the development of the HCC. In this case, the challenges are (1 there are few markers specific to the HCC (tumor cells versus nontumor cells and (2 they can be used to quantify the number of CTC in the bloodstream. Another technical challenge consists of finding few CTC mixed with million leukocytes and billion erythrocytes. CTC detection and identification can be used to estimate prognosis and may serve as an early marker to assess antitumor activity of treatment. CTC can also be used to predict progression-free survival and overall survival. CTC are an interesting source of biological information in order to understand dissemination, drug resistance, and treatment-induced cell death. Our aim is to review and analyze the different new methods existing to detect, enumerate, and characterize the CTC in the peripheral circulation of patients with HCC.

  13. Prognostic Importance of Circulating Tumor Cells in Nonsmall Cell ...

    African Journals Online (AJOL)

    Purpose: To investigate the prognostic value of circulating tumor cells (CTCs) and to predict the treatment response in a non-small cell lung cancer (NSCLC). Methodology: A single-center prospective study involving 93 patients with NSCLC was conducted. Blood samples were analyzed for CTC count before and after ...

  14. Circulating tumor cells in melanoma patients.

    Directory of Open Access Journals (Sweden)

    Gary A Clawson

    Full Text Available Circulating tumor cells (CTCs are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X from blood of melanoma patients using a simple centrifugation device (OncoQuick, and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001. There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001, and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50% stained for both pan-cytokeratin (KRT markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14. Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs. The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids

  15. Circulating tumor cells: utopia or reality?

    Science.gov (United States)

    Conteduca, Vincenza; Zamarchi, Rita; Rossi, Elisabetta; Condelli, Valentina; Troiani, Laura; Aieta, Michele

    2013-09-01

    Circulating tumor cells (CTCs) could be considered a sign of tumor aggressiveness, but highly sensitive and specific methods of CTC detection are necessary owing to the rarity and heterogeneity of CTCs in peripheral blood. This review summarizes recent studies on tumor biology, with particular attention to the metastatic cascade, and the molecular characterization and clinical significance of CTCs. Recent technological approaches to enrich and detect these cells and challenges of CTCs for individualized cancer treatment are also discussed. This review also provides an insight into the positive and negative features of the future potential applications of CTC detection, which sometimes remains still a 'utopia', but its actual utility remains among the fastest growing research fields in oncology.

  16. Electrical Detection Method for Circulating Tumor Cells Using Graphene Nanoplates.

    Science.gov (United States)

    Han, Song-I; Han, Ki-Ho

    2015-10-20

    This paper presents a microfluidic device for electrical discrimination of circulating tumor cells (CTCs) using graphene nanoplates (GNPs) as a highly conductive material bound to the cell surface. For two-step cascade discrimination, the microfluidic device is composed of a CTC-enrichment device and an impedance cytometry. Using lateral magnetophoresis, the CTC-enrichment device enriches rare CTCs from millions of background blood cells. Then, the impedance cytometry electrically identifies CTCs from the enriched sample, containing CTCs and persistent residual blood cells, based on the electrical impedance of CTCs modified by the GNPs. GNPs were used as a highly conductive material for modifying surface conductivity of CTCs, thereby improving the accuracy of electrical discrimination. The experimental results showed that a colorectal cancer cell line (DLD-1) spiked into peripheral blood was enriched by nearly 500-fold by the CTC-enrichment device. The phase of the electrical signal measured from DLD-1 cells covered by GNPs shifted by about 100° in comparison with that from normal blood cells, which allows the impedance cytometry to identify CTCs at a rate of 94% from the enriched samples.

  17. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer.

    Science.gov (United States)

    Riethdorf, Sabine; Soave, Armin; Rink, Michael

    2017-12-01

    Urothelial carcinoma of the bladder (UCB) is a complex disease, which is associated with highly aggressive tumor biologic behavior, especially in patients with muscle-invasive and advanced tumors. Despite multimodal therapy options including surgery, radiotherapy and chemotherapy, UCB patients frequently suffer from poor clinical outcome. Indeed, the potential of diverse opportunities for modern targeted therapies is not sufficiently elucidated in UCB yet. To improve the suboptimal treatment situation in UCB, biomarkers are urgently needed that help detecting minimal residual disease (MRD), predicting therapy response and subsequently prognosis as well as enabling patient stratification for further therapies and therapy monitoring, respectively. To date, decision making regarding treatment planning is mainly based on histopathologic evaluation of biopsies predominantly derived from the primary tumors and on clinical staging. However, both methods are imperfect for sufficient outcome prediction. During disease progression, individual disseminated tumor cells and consecutively metastases can acquire characteristics that do not match those of the corresponding primary tumors, and often are only hardly assessable for further evaluation. Therefore, during recent years, strong efforts were directed to establish non-invasive biomarkers from liquid biopsies. Urine cytology and serum tumor markers have been established for diagnostic purposes, but are still insufficient as universal biomarkers for decision-making and treatment of UCB patients. To date, the clinical relevance of various newly established blood-based biomarkers comprising circulating tumor cells (CTCs), circulating cell-free nucleic acids or tumor-educated platelets is being tested in cancer patients. In this review we summarize the current state and clinical application of CTCs and circulating cell-free tumor DNA originating from blood as biomarkers in patients with different UCB stages.

  18. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer

    Science.gov (United States)

    Soave, Armin; Rink, Michael

    2017-01-01

    Urothelial carcinoma of the bladder (UCB) is a complex disease, which is associated with highly aggressive tumor biologic behavior, especially in patients with muscle-invasive and advanced tumors. Despite multimodal therapy options including surgery, radiotherapy and chemotherapy, UCB patients frequently suffer from poor clinical outcome. Indeed, the potential of diverse opportunities for modern targeted therapies is not sufficiently elucidated in UCB yet. To improve the suboptimal treatment situation in UCB, biomarkers are urgently needed that help detecting minimal residual disease (MRD), predicting therapy response and subsequently prognosis as well as enabling patient stratification for further therapies and therapy monitoring, respectively. To date, decision making regarding treatment planning is mainly based on histopathologic evaluation of biopsies predominantly derived from the primary tumors and on clinical staging. However, both methods are imperfect for sufficient outcome prediction. During disease progression, individual disseminated tumor cells and consecutively metastases can acquire characteristics that do not match those of the corresponding primary tumors, and often are only hardly assessable for further evaluation. Therefore, during recent years, strong efforts were directed to establish non-invasive biomarkers from liquid biopsies. Urine cytology and serum tumor markers have been established for diagnostic purposes, but are still insufficient as universal biomarkers for decision-making and treatment of UCB patients. To date, the clinical relevance of various newly established blood-based biomarkers comprising circulating tumor cells (CTCs), circulating cell-free nucleic acids or tumor-educated platelets is being tested in cancer patients. In this review we summarize the current state and clinical application of CTCs and circulating cell-free tumor DNA originating from blood as biomarkers in patients with different UCB stages. PMID:29354496

  19. Isolation of Circulating Tumor Cells by Dielectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Gascoyne, Peter R. C., E-mail: pgascoyn@mdanderson.org [Department of Imaging Physics Research, The University of Texas M.D. Anderson Cancer Center Unit 951, 1515 Holcombe Boulevard, Houston, TX 77030 (United States); Shim, Sangjo [Department of Imaging Physics Research, The University of Texas M.D. Anderson Cancer Center Unit 951, 1515 Holcombe Boulevard, Houston, TX 77030 (United States); Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, C0800, Austin, TX 78712 (United States); Present address: Micro & Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, 208 North Wright Street, Urbana, IL 61801 (United States)

    2014-03-12

    Dielectrophoresis (DEP) is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs) from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a) the principles of DEP; (b) the biological basis for the dielectric differences between CTCs and blood cells; (c) why such differences are expected to be present for all types of tumors; and (d) instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF) is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies.

  20. Isolation of Circulating Tumor Cells by Dielectrophoresis

    Directory of Open Access Journals (Sweden)

    Peter R. C. Gascoyne

    2014-03-01

    Full Text Available Dielectrophoresis (DEP is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a the principles of DEP; (b the biological basis for the dielectric differences between CTCs and blood cells; (c why such differences are expected to be present for all types of tumors; and (d instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies.

  1. Circulating Tumor Cells, Enumeration and Beyond

    Directory of Open Access Journals (Sweden)

    Jian-Mei Hou

    2010-06-01

    Full Text Available The detection and enumeration of circulating tumor cells (CTCs has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a ‘virtual and real-time biopsy’ have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial ‘needle in the haystack’. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology.

  2. Circulating Tumor Cells, Enumeration and Beyond

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Jian-Mei [Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester M20 4BX (United Kingdom); Krebs, Matthew [Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester M20 4BX (United Kingdom); Christie Hospital Foundation NHS Trust, Manchester M20 4BX (United Kingdom); Ward, Tim; Morris, Karen; Sloane, Robert [Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester M20 4BX (United Kingdom); Blackhall, Fiona [Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester M20 4BX (United Kingdom); School of Cancer and Enabling Sciences, University of Manchester, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, Manchester M20 4BX (United Kingdom); Christie Hospital Foundation NHS Trust, Manchester M20 4BX (United Kingdom); Dive, Caroline, E-mail: cdive@picr.man.ac.uk [Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester M20 4BX (United Kingdom); School of Cancer and Enabling Sciences, University of Manchester, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, Manchester M20 4BX (United Kingdom)

    2010-06-09

    The detection and enumeration of circulating tumor cells (CTCs) has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a ‘virtual and real-time biopsy’ have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial ‘needle in the haystack’. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology.

  3. Circulating Tumor Cells, Enumeration and Beyond

    International Nuclear Information System (INIS)

    Hou, Jian-Mei; Krebs, Matthew; Ward, Tim; Morris, Karen; Sloane, Robert; Blackhall, Fiona; Dive, Caroline

    2010-01-01

    The detection and enumeration of circulating tumor cells (CTCs) has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a ‘virtual and real-time biopsy’ have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial ‘needle in the haystack’. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology

  4. Circulating osteogenic cells: implications for injury, repair, and regeneration

    DEFF Research Database (Denmark)

    Pignolo, Robert J; Kassem, Moustapha

    2011-01-01

    The aim of this review is to provide a critical reading of recent literature pertaining to the presence of circulating, fluid-phase osteoblastic cells and their possible contribution to bone formation. We have termed this group of cells collectively as circulating osteogenic precursor (COP) cells...

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

    Science.gov (United States)

    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.

  6. Leaching Behavior of Circulating Fluidised Bed MSWI Air Pollution Control Residue in Washing Process

    Directory of Open Access Journals (Sweden)

    Zhiliang Chen

    2016-09-01

    Full Text Available In this study, air pollution control (APC residue is conducted with water washing process to reduce its chloride content. A novel electrical conductivily (EC measurement method is proposed to monitor the dynamic change of chloride concentrations in leachate as well as the chloride content of the residue. The method equally applies to various washing processes with different washing time, liquid/solid ratio and washing frequency. The results show that washing effectively extracts chloride salts from APC residues, including those from circulating fluidized bed (CFB municipal solid waste incineration (MSWI. The most appropriate liquid/solid ratio and washing time in the first washing are found to be around 4 L water per kg of APC residue and 30 min, respectively, and washing twice is required to obtain maximum dissolution. The pH value is the major controlling factor of the heavy metals speciation in leachate, while chloride concentration also affects the speciation of Cd. Water washing causes no perceptible transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs from the APC residue to leachate. The chloride concentration is strongly related with electrical conductivity (EC, as well as with the concentrations of calcium, sodium and potassium of washing water. Their regression analyses specify that soluble chloride salts and EC could act as an indirect indicator to monitor the change of chloride concentration and remaining chloride content, thus, contributing to the selection of the optimal washing conditions.

  7. Molecular biomarker analyses using circulating tumor cells.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Punnoose

    2010-09-01

    Full Text Available Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs isolated from blood of metastatic cancer patients hold significant promise in this regard.Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF. We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89% we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%, HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC

  8. Microfluidic Platform for Circulating Tumor Cells Isolation

    Energy Technology Data Exchange (ETDEWEB)

    Figueras-Mari, I.; Rodriguez-Trujillo, L.; Samitier-Marti, J.

    2016-07-01

    Circulating tumor cells (CTCs) are released from primary tumors into the bloodstream and transported to distant organs, promoting metastasis, which is known to be responsible for most cancer‐related deaths. Currently tumors are not found until symptoms appear or by chance when the patient undergoes a medical test, which in both situations can be too late. Once a tumor is found it is studied from tissue samples obtained directly from the patient in an invasive way. This invasive procedure is known as biopsy and apart from being invasive, it is costly, time consuming and can sometimes be painful and even risky for the patients’ health condition. Therefore, CTCs detection in blood also addressed as “liquid biopsy” would be very useful because by running routine blood analysis CTCs could be detected and collected suggesting tumor presence. However, due to the scarce presence in blood of these cells and to the huge amount of contamination from other cellular components a perfect method providing good capture and purity of CTCs has not been developed yet. In this project, a spiral size sorter microfluidic device has been manufactured and tested in order to determine its performance and limitations. Device performance was tested with different dilutions of healthy donor blood samples mixed with 30 micron particles simulating CTCs. The results obtained from these experiments show very good CTC recovery of up to 100% and the depletion of blood cellular components is around 99.9%. (Author)

  9. Extracorporeal Photo-Immunotherapy for Circulating Tumor Cells

    OpenAIRE

    Kim, Gwangseong; Gaitas, Angelo

    2015-01-01

    It is well established that metastasis through the circulatory system is primarily caused by circulating tumor cells (CTCs). In this preliminary effort, we report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT). Chlorin e6 (Ce6), a photosensitizer, was conjugated to CD44 antibody in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-...

  10. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and ...

  11. Characterizing T Cells in SCID Patients Presenting with Reactive or Residual T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Atar Lev

    2012-01-01

    Full Text Available Introduction. Patients with severe combined immunodeficiency (SCID may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Methods. Here we compared T-cell functions including the number of circulating CD3+ T cells, in vitro responses to mitogens, T-cell receptor (TCR repertoire, TCR excision circles (TREC levels, and regulatory T cells (Tregs enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

  12. Circulating cell free DNA as a predictor of systemic lupus ...

    African Journals Online (AJOL)

    Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy. Olfat M. Hendy, Tawfik Abdel Motalib, Mona A. El Shafie, Fatma A. Khalaf, Sobhy E. Kotb, Aziza Khalil, Salwa R. Ali ...

  13. Estimated residual Magnetic Field acting on the Circulating Beam in the LHC Septum Magnets MSI and MSD - Shielding Efficiency

    CERN Document Server

    Gyr, Marcel

    2000-01-01

    Computation of the residual field inside the shielded vacuum chambers of the circulating beams that pass through the septum holes of the MSI and MSD magnets is very impractical because of the disproportionate effort involved in obtaining meaningful results. Therefore, a short model has been built to measure the order of magnitude of the residual field inside the LHC vacuum chambers. It is found, that a 0.9 mm thick µ-metal (or Permalloy$^{TM}$) shielding is sufficient to reduce the field, which is experienced by the circulating beam, below the level of the earth's field.

  14. Circulating melanoma cells and survival in metastatic melanoma

    NARCIS (Netherlands)

    Rao, C.; Bui, T.; Connelly, M.; Doyle, G.; Karydis, I.; Middleton, M. R.; Clack, G.; Malone, M.; Coumans, F. A. W.; Terstappen, L. W. M. M.

    2011-01-01

    A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146(+) cells were immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with

  15. Innovative Microsystems: Novel Nanostructures to Capture Circulating Breast Cancer Cells

    Science.gov (United States)

    2009-05-01

    The goal of this project is to develop a microsystem for sorting metastatic breast cancer cells from a heterogeneous suspension of cells circulating...expression vector deriving a homogeneous population. An anti-N-cad functionalized surface has been shown to capture N-cad expressing prostate cancer cells (PC3N

  16. Clusters of Circulating Tumor Cells: a Biophysical and Technological Perspective.

    Science.gov (United States)

    Au, Sam H; Edd, Jon; Haber, Daniel A; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet

    2017-09-01

    The vast majority of cancer associated deaths result from metastasis, yet the behaviors of its most potent cellular driver, circulating tumor cell clusters, are only beginning to be revealed. This review highlights recent advances to our understanding of tumor cell clusters with emphasis on enabling technologies. The importance of intercellular adhesions among cells in clusters have begun to be unraveled with the aid of promising microfluidic strategies for isolating clusters from patient blood. Due to their metastatic potency, the utility of circulating tumor cell clusters for cancer diagnosis, drug screening, precision oncology and as targets of antimetastatic therapeutics are being explored. The continued development of tools for exploring circulating tumor cell clusters will enhance our fundamental understanding of the metastatic process and may be instrumental in devising new strategies to suppress and eliminate metastasis.

  17. Circulating mesenchymal stem cells and their clinical implications

    Directory of Open Access Journals (Sweden)

    Liangliang Xu

    2014-01-01

    Full Text Available Circulating mesenchymal stem cells (MSCs is a new cell source for tissue regeneration and tissue engineering. The characteristics of circulating MSCs are similar to those of bone marrow-derived MSCs (BM-MSCs, but they exist at a very low level in healthy individuals. It has been demonstrated that MSCs are able to migrate to the sites of injury and that they have some distinct genetic profiles compared to BM-MSCs. The current review summaries the basic knowledge of circulating MSCs and their potential clinical applications, such as mobilizing the BM-MSCs into circulation for therapy. The application of MSCs to cure a broad spectrum of diseases is promising, such as spinal cord injury, cardiovascular repair, bone and cartilage repair. The current review also discusses the issues of using of allogeneic MSCs for clinical therapy.

  18. Filter characteristics influencing circulating tumor cell enrichment from whole blood.

    NARCIS (Netherlands)

    Coumans, F.A.W.; van Dalum, Guus; Beck, Markus; Terstappen, Leonardus Wendelinus Mathias Marie

    2013-01-01

    A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of

  19. Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

    NARCIS (Netherlands)

    Coumans, Frank A. W.; van Dalum, Guus; Beck, Markus; Terstappen, Leon W. M. M.

    2013-01-01

    A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of

  20. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  1. Circulating immature osteoprogenitor cells and arterial stiffening in postmenopausal osteoporosis.

    Science.gov (United States)

    Pirro, M; Schillaci, G; Mannarino, M R; Scarponi, A M; Manfredelli, M R; Callarelli, L; Leli, C; Fabbriciani, G; Helou, R S; Bagaglia, F; Mannarino, E

    2011-09-01

    An increased number of circulating osteoprogenitor cells (OPCs) expressing bone-related proteins and the stem cell marker CD34 have been identified in women with postmenopausal osteoporosis, who also have stiffer arteries than nonosteoporotic subjects. We investigated whether an increased number of circulating OPCs underlies the association of osteoporosis with arterial stiffness. The number of circulating OPCs was quantified by FACS analysis in 120 postmenopausal women with or without osteoporosis. OPCs were defined as CD34+/alkaline phosphatase(AP)+ or CD34+/osteocalcin(OCN)+ cells. Participants underwent cardiovascular risk factor assessment, measurement of bone mineral density (BMD), and aortic pulse wave velocity (aPWV) as a measure of arterial stiffness. Osteoporotic women had higher aPWV (9.8 ± 2.8 vs 8.5 ± 1.9 m/s, p = 0.005) and levels of CD34+/AP+ and CD34+/OCN+ cells than nonosteoporotic controls [1045 n/mL (487-2300) vs 510 n/mL (202-940), p osteoporosis an increased availability of circulating osteoprogenitor cells has a detrimental influence on arterial compliance, which may in part explain the association between osteoporosis and arterial stiffening. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer

    NARCIS (Netherlands)

    Pore, M.M.; Meijer, C.; de Bock, G.H.; Boersma-van Ek, W.; Terstappen, Leonardus Wendelinus Mathias Marie; Groen, H.J.M.; Timens, W.; Kruyt, F.A.E.; Hiltermann, T.N.J.

    2016-01-01

    Background Small cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and

  3. Extracorporeal photo-immunotherapy for circulating tumor cells.

    Directory of Open Access Journals (Sweden)

    Gwangseong Kim

    Full Text Available It is well established that metastasis through the circulatory system is primarily caused by circulating tumor cells (CTCs. In this preliminary effort, we report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT. Chlorin e6 (Ce6, a photosensitizer, was conjugated to CD44 antibody in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-CD44 antibody conjugate. PDT was performed on whole blood spiked with stained PC-3 cells. As the blood circulated through a thin transparent medical tube, it was exposed to light of 660 nm wavelength generated by an LED array. An exposure of two minutes was sufficient to achieve selective cancer cell necrosis. In comparison, to PDT of cells growing inside a tissue culture, the PDT on thin tube exhibited significantly enhanced efficiency in cell killing, by minimizing light attenuation by blood. It suggests a new extracorporeal methodology of PDT for treating CTCs as well as other hematological pathogens.

  4. Extracorporeal photo-immunotherapy for circulating tumor cells.

    Science.gov (United States)

    Kim, Gwangseong; Gaitas, Angelo

    2015-01-01

    It is well established that metastasis through the circulatory system is primarily caused by circulating tumor cells (CTCs). In this preliminary effort, we report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT). Chlorin e6 (Ce6), a photosensitizer, was conjugated to CD44 antibody in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-CD44 antibody conjugate. PDT was performed on whole blood spiked with stained PC-3 cells. As the blood circulated through a thin transparent medical tube, it was exposed to light of 660 nm wavelength generated by an LED array. An exposure of two minutes was sufficient to achieve selective cancer cell necrosis. In comparison, to PDT of cells growing inside a tissue culture, the PDT on thin tube exhibited significantly enhanced efficiency in cell killing, by minimizing light attenuation by blood. It suggests a new extracorporeal methodology of PDT for treating CTCs as well as other hematological pathogens.

  5. Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization

    DEFF Research Database (Denmark)

    Nejlund, Sarah; Smith, Julie; Kraan, Jaco

    2016-01-01

    BACKGROUND: A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of exi...

  6. Circulating dendritic cells in pediatric patients with nephrotic syndrome

    African Journals Online (AJOL)

    Background: Dendritic cells (DCs) represent one of the most extensively studied topics in immunology, because of their central role in the induction and regulation of adaptive immunity, and because of their therapeutic potential for manipulating immune responses. Objectives: To evaluate circulating DC levels in pediatric ...

  7. Donor-derived circulating endothelial cells after kidney transplantation

    NARCIS (Netherlands)

    Popa, ER; Kas-Deelen, AM; Hepkema, BG; van Son, WJ; The, TH; Harmsen, MC

    2002-01-01

    Background. In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of

  8. Expression profiling of circulating tumor cells in metastatic breast cancer

    Czech Academy of Sciences Publication Activity Database

    Lang, J.; Scott, J.H.; Wolf, D.M.; Novák, Petr; Punj, V.; Magbanua, M.J.M.; Zhu, W.Z.; Mineyev, N.; Haqq, CH.; Crothers, J.

    2015-01-01

    Roč. 149, č. 1 (2015), s. 121-131 ISSN 0167-6806 Institutional support: RVO:60077344 Keywords : Circulating tumor cells * Micrometastases * Breast cancer * EpCAM Subject RIV: FD - Oncology ; Hematology Impact factor: 4.085, year: 2015

  9. Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons.

    Science.gov (United States)

    Tan, Carlyn Rose C; Zhou, Lanlan; El-Deiry, Wafik S

    2016-06-01

    Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are emerging noninvasive multifunctional biomarkers in liquid biopsy allowing for early diagnosis, accurate prognosis, therapeutic target selection, spatiotemporal monitoring of metastasis, as well as monitoring response and resistance to treatment. CTCs and ctDNA are released from different tumor types at different stages and contribute complementary information for clinical decision. Although big strides have been taken in technology development for detection, isolation and characterization of CTCs and sensitive and specific detection of ctDNA, CTC-, and ctDNA-based liquid biopsies may not be widely adopted for routine cancer patient care until the suitability, accuracy, and reliability of these tests are validated and more standardized protocols are corroborated in large, independent, prospectively designed trials. This review covers CTC- and ctDNA-related technologies and their application in colorectal cancer. The promise of CTC-and ctDNA-based liquid biopsies is envisioned.

  10. Circulating granulocytic and erythroid progenitor cells in chronic granulocytic leukaemia.

    Science.gov (United States)

    Goldman, J M; Shiota, F; Th'ng, K H; Orchard, K H

    1980-09-01

    We used a standard methyl cellulose method to assay erythroid progenitor cells in the blood of 35 patients with untreated CGL and of 18 normal controls. In 28 patients we simultaneously assayed granulocyte/monocyte committed progenitor cells (CFU-c) by an agar method. Circulating erythroid burst-forming units (BFU-e) in CGL were increased above normal by a factor of about 180; CFU-c were increased by a factor of about 9000. Both BFU-e and CFU-c numbers were linearly related to the total leucocyte count in individual patients but not to numbers of circulating blast cells. There was a positive correlation in individual patients between CFU-c and BFU-e numbers. Circulating BFU-e and erythroid colony-forming cells (CFU-e) were unable to proliferate in vitro in the absence of erythropoietin. We conclude that erythroid progenitor cells are involved in the 'clonal expansion' that characterizes CGL, but apparently to a lesser extent than are granulocyte/moonocyte progenitor cells.

  11. Photoacoustic imaging of single circulating melanoma cells in vivo

    Science.gov (United States)

    Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

    2015-03-01

    Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

  12. Isolation and characterization of circulating tumor cells in prostate cancer

    Directory of Open Access Journals (Sweden)

    Elan Shlomo Diamond

    2012-10-01

    Full Text Available Circulating tumor cells (CTCs are tumor cells found in the peripheral blood that originate from established sites of malignancy and likely have metastatic potential. Analysis of circulating tumor cells CTCs has shown great promise as a prognostic marker as well as a potential source of novel therapeutics. Isolation and characterization these cells for study, however, remain challenging due to their rarity in comparison with other cellular components of peripheral blood. Several techniques that exploit the unique biochemical properties of CTCs have been developed to facilitate isolation of these cells. Positive selection of CTCs is achieved using microfluidic surfaces coated with antibodies against epithelial cell markers or tumor specific antigens such as EpCAM or prostate specific membrane antigen (PSMA. Following isolation, characterization of CTCs may help guide clinical decision-making. For instance, molecular and genetic characterization may shed light on the development of chemotherapy resistance and mechanisms of metastasis without the need for tissue biopsy. This paper will review novel isolation techniques to capture CTCs from patients with advanced cancers, as well as efforts to characterize the CTCs. We will also review ways in which these analyses can assist in clinical decision-making,Conclusion: The study of CTCs provides insight into the molecular biology of their tumors of origin that will eventually guide the development tailored therapeutics. These advances are predicated on high yield and accurate isolation techniques that exploit the unique biochemical features of these cells.

  13. Circulating endothelial progenitor cells in obese children and adolescents.

    Science.gov (United States)

    Pires, António; Martins, Paula; Paiva, Artur; Pereira, Ana Margarida; Marques, Margarida; Castela, Eduardo; Sena, Cristina; Seiça, Raquel

    2015-01-01

    This study aimed to investigate the relationship between circulating endothelial progenitor cell count and endothelial activation in a pediatric population with obesity. Observational and transversal study, including 120 children and adolescents with primary obesity of both sexes, aged 6-17 years, who were recruited at this Cardiovascular Risk Clinic. The control group was made up of 41 children and adolescents with normal body mass index. The variables analyzed were: age, gender, body mass index, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance, monocyte chemoattractant protein-1, E-selectin, asymmetric dimethylarginine and circulating progenitor endothelial cell count. Insulin resistance was correlated to asymmetric dimethylarginine (ρ=0.340; p=0.003), which was directly, but weakly correlated to E-selectin (ρ=0.252; p=0.046). High sensitivity C-reactive protein was not found to be correlated to markers of endothelial activation. Systolic blood pressure was directly correlated to body mass index (ρ=0.471; p<0.001) and the homeostasis model assessment-insulin resistance (ρ=0.230; p=0.012), and inversely correlated to adiponectin (ρ=-0.331; p<0.001) and high-density lipoprotein cholesterol (ρ=-0.319; p<0.001). Circulating endothelial progenitor cell count was directly, but weakly correlated, to body mass index (r=0.211; p=0.016), leptin (ρ=0.245; p=0.006), triglyceride levels (r=0.241; p=0.031), and E-selectin (ρ=0.297; p=0.004). Circulating endothelial progenitor cell count is elevated in obese children and adolescents with evidence of endothelial activation, suggesting that, during infancy, endothelial repairing mechanisms are present in the context of endothelial activation. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  14. Specific recruitment of circulating angiogenic cells using biomaterials as filters.

    Science.gov (United States)

    Parlato, Matthew; Molenda, James; Murphy, William L

    2017-07-01

    Endogenous recruitment of circulating angiogenic cells (CACs) is an emerging strategy to induce angiogenesis within a defect site, and multiple recent strategies have deployed soluble protein releasing biomaterials for this purpose. However, the way in which the design of biomaterials affects CAC recruitment and invasion are poorly understood. Here we used an enhanced-throughput cell invasion assay to systematically examine the effects of biomaterial design on CAC recruitment. The screens co-optimized hydrogel presentation of a stromal-derived factor-1α (SDF-1α) gradient, hydrogel degradability, and hydrogel stiffness for maximal CAC invasion. We also examined the specificity of this invasion by assessing dermal fibroblast, mesenchymal stem cell, and lymphocyte invasion individually and in co-culture with CACs to identify hydrogels specific to CAC invasion. These screens suggested a subset of MMP-degradable hydrogels presenting a specific range of SDF-1α gradient slopes that induced specific invasion of CACs, and we posit that the design parameters of this subset of hydrogels may serve as instructive templates for the future design of biomaterials to specifically recruit CACs. We also posit that this design concept may be applied more broadly in that it may be possible to utilize these specific subsets of biomaterials as "filters" to control which types of cell populations invade into and populate the biomaterial. The recruitment of specific cell types for cell-based therapies in vivo is of great interest to the regenerative medicine community. Circulating angiogenic cells (CACs), CD133+ cells derived from the blood stream, are of particular interest for induction of angiogenesis in ischemic tissues, and recent studies utilizing soluble-factor releasing biomaterials to recruit these cells in vivo show great promise. However, these studies are largely "proof of concept" and are not systematic in nature. Thus, little is currently known about how biomaterial design

  15. Circulating tumor DNA outperforms circulating tumor cells for KRAS mutation detection in thoracic malignancies.

    Science.gov (United States)

    Freidin, Maxim B; Freydina, Dasha V; Leung, Maria; Montero Fernandez, Angeles; Nicholson, Andrew G; Lim, Eric

    2015-10-01

    Circulating biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are both considered for blood-based mutation detection, but limited studies have compared them in a head-to-head manner. Using KRAS (Kirsten rat sarcoma viral oncogene homolog), we performed such a comparison in patients who underwent surgery for suspected lung cancer. We recruited 93 patients, including 82 with lung cancer and 11 with benign diseases of the lung. Mutations were detected in codons 12 and 13 of KRAS in DNA extracted from CTCs, plasma, and matched tumors or lung tissues with custom-designed coamplification at lower denaturation temperature (COLD)-PCR assays, high-resolution melt analysis (HRM), and commercial assays (Roche Cobas(®) KRAS mutation test and Qiagen Therascreen(®) pyrosequencing KRAS kit). With the Cobas mutation test, we identified KRAS mutations in 21.3% of tumors. Mutation analysis in matched CTC DNA and ctDNA samples by COLD-PCR/HRM assay revealed mutations in 30.5% (ctDNA) and 23.2% (CTC DNA) of patients with lung cancer. Combined results of different tests revealed KRAS-positive cases for 28% of tumors. The diagnostic sensitivity and specificity of KRAS mutation detection in tumors achieved with ctDNA was 0.96 (95% CI 0.81-1.00) and 0.95 (0.85-0.99), respectively. The diagnostic test performance was lower for CTC DNA, at 0.52 (0.34-0.73) and 0.88 (0.79-0.95). Our results support ctDNA as a preferential specimen type for mutation screening in thoracic malignancies vs CTC DNA, achieving greater mutation detection than either CTCs or limited amounts of tumor tissue alone. © 2015 American Association for Clinical Chemistry.

  16. Circulating tumor cells: the substrate of personalized medicine?

    Directory of Open Access Journals (Sweden)

    Bryan eGreene

    2012-07-01

    Full Text Available Circulating tumor cells (CTCs are believed to be responsible for the development of metastatic disease. Over the last several years there has been a great interest in understanding the biology of CTCs to understand metastasis, as well as for the development of companion diagnostics to predict patient response to anti-cancer targeted therapies. Understanding CTC biology requires innovative technologies for the isolation of these rare cells. Here we review several methods for the detection, capture, and analysis of CTCs and also provide insight on improvements for CTC capture amenable to cellular therapy applications.

  17. Circulating Tumor Cells and Circulating Tumor DNA Provide New Insights into Pancreatic Cancer.

    Science.gov (United States)

    Gao, Yang; Zhu, Yayun; Yuan, Zhou

    2016-01-01

    Pancreatic cancer has a rather dismal prognosis mainly due to high malignance of tumor biology. Up to now, the relevant researches on pancreatic cancer lag behind seriously partly due to the obstacles for tissue biopsy, which handicaps the understanding of molecular and genetic features of pancreatic cancer. In the last two decades, liquid biopsy, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), is promising to provide new insights into the biological and clinical characteristics of malignant tumors. Both CTCs and ctDNA provide an opportunity for studying tumor heterogeneity, drug resistance, and metastatic mechanism for pancreatic cancer. Furthermore, they can also play important roles in detecting early-stage tumors, providing prognostic information, monitoring tumor progression and guiding treatment regimens. In this review, we will introduce the latest findings on biological features and clinical applications of both CTCs and ctDNA in pancreatic cancer. In a word, CTCs and ctDNA are promising to promote precision medicine in pancreatic cancer.

  18. Foreign fetal cells persist in the maternal circulation.

    Science.gov (United States)

    Williams, Zev; Zepf, Dimity; Longtine, Janina; Anchan, Raymond; Broadman, Betsy; Missmer, Stacey A; Hornstein, Mark D

    2009-06-01

    To determine whether allogenic fetal cells resulting from donor egg pregnancies persist in maternal circulation. Nested polymerase chain reaction (PCR) amplification of the DYS14 sequence, a region of the Y chromosome, from DNA purified from peripheral blood cells. Academic medical center. Healthy 18-60-year-old women who have had donor egg pregnancies resulting in a male offspring (n = 11) or, as a control, female offspring (n = 8), at least 1 year previously and without any other source for male cells in their peripheral blood or a healthy male. None. Detection of DYS14 sequence by nested PCR. DYS14 was detected in 5/11 (45%) of women who had donor egg pregnancies resulting in a male offspring, but in 0/8 (0) of women who had donor egg pregnancies resulting in a female offspring. The longest interval between delivery of a male offspring and detection of the DYS14 gene was 9 years. Unmatched, allogenic fetal cells from donor egg pregnancies are able to persist in the circulation of healthy women for at least 9 years after delivery. This implies a novel mechanism by which immunologic detection is avoided by these cells and may impact on how they may be used for regenerative and transplant medicine.

  19. Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

    Science.gov (United States)

    Beck, Markus; Terstappen, Leon W. M. M.

    2013-01-01

    Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104∶102∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC. PMID:23658615

  20. Filtration parameters influencing circulating tumor cell enrichment from whole blood.

    Directory of Open Access Journals (Sweden)

    Frank A W Coumans

    Full Text Available Filtration can achieve circulating tumor cell (CTC enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10(4∶10(2∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm(2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

  1. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    Science.gov (United States)

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  2. Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients

    Czech Academy of Sciences Publication Activity Database

    Hensler, M.; Vancurova, I.; Becht, E.; Palata, O.; Strnad, P.; Tesarova, P.; Cabinakova, M.; Švec, David; Kubista, Mikael; Bartunkova, J.; Spisek, R.; Sojka, L.

    2016-01-01

    Roč. 5, č. 4 (2016), e1102827 ISSN 2162-402X Institutional support: RVO:86652036 Keywords : Breast cancer * gene expression profiling * circulating tumor cells Subject RIV: FD - Oncology ; Hematology Impact factor: 7.719, year: 2016

  3. Residual circulation and thermohaline distribution of the Ría de Vigo: A 3-D hydrodynamical model

    Directory of Open Access Journals (Sweden)

    S. Torres López

    2001-07-01

    Full Text Available A three-dimensional, non-linear, baroclinic model is described and tested for the first time to study the residual circulation and the thermohaline distribution of the Ría de Vigo (NW Spain at short time scales and under different wind stress regimes. Two markedly different realistic scenarios were chosen: northerly upwelling-favourable winds and southerly downwelling-favourable winds. The numerical experiments carried out indicate that the hydrodynamic regime of the Ría de Vigo is mostly a consequence of wind events. As could be expected, moderately strong North winds reinforce the normal (positive Ría circulation, while winds blowing from the South, when sufficiently strong, reverse the typical circulation pattern and reduce the characteristic outgoing velocities and the flushing time inside the Ría. The temperature and salinity fields generated by the model in both situations were compared with observations and found to be in qualitatively good agreement, supporting the 3D velocity field distribution.

  4. Single Cell Characterization of Prostate Cancer-Circulating Tumor Cells

    Science.gov (United States)

    2013-09-01

    contaminating WBC. Scale bar = 20 microns. (E) MagSweeper versus CellSearch comparison. Samples with 0 CTC were assigned a value of 1. 10...cancer patient blood sample, and contaminating WBC found after MagSweeper isolation. Scale bar = 20 microns. (E) MagSweeper versus CellSearch...Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 4. Ashworth TR (1869) A case of cancer in which cells similar to those in

  5. Filter characteristics influencing circulating tumor cell enrichment from whole blood.

    Directory of Open Access Journals (Sweden)

    Frank A W Coumans

    Full Text Available A variety of filters assays have been described to enrich circulating tumor cells (CTC based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45-DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9-19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11-13 µm should be used to challenge the system.

  6. Circulating endothelial cells: a potential parameter of organ damage in sickle cell anemia?

    NARCIS (Netherlands)

    Strijbos, Michiel H.; Landburg, Precious P.; Nur, Erfan; Teerlink, Tom; Leebeek, Frank W. G.; Rijneveld, Anita W.; Biemond, Bart J.; Sleijfer, Stefan; Gratama, Jan W.; Duits, Ashley J.; Schnog, John-John B.

    2009-01-01

    Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count

  7. The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yuji Mishima

    2017-04-01

    Full Text Available The development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs is prognostic in multiple myeloma (MM, but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.

  8. The Potential for Circulating Tumor Cells in Pancreatic Cancer Management

    Directory of Open Access Journals (Sweden)

    Michael Pimienta

    2017-06-01

    Full Text Available Pancreatic cancer is one the most lethal malignancies. Only a small proportion of patients with this disease benefit from surgery. Chemotherapy provides only a transient benefit. Though much effort has gone into finding new ways for early diagnosis and treatment, average patient survival has only been improved in the order of months. Circulating tumor cells (CTCs are shed from primary tumors, including pre-malignant phases. These cells possess information about the genomic characteristics of their tumor source in situ, and their detection and characterization holds potential in early cancer diagnosis, prognosis, and treatment. Liquid Biopsies present an alternative to tumor biopsy that are hard to sample. Below we summarize current methods of CTC detection, the current literature on CTCs in pancreatic cancer, and future perspectives.

  9. Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.

    Science.gov (United States)

    Wang, Denong; Wu, Lisa; Liu, Xiaohe

    2017-01-01

    We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

  10. Circulating cells as biomarkers in cardiovascular disease : the difference between men and women

    NARCIS (Netherlands)

    Zweers, Thijs

    2015-01-01

    During this research project we studied circulating cells in the blood of people with cardiovascular disease, we investigated if these cells could be used as biomarkers for future cardiovascular incidents. We specifically looked at circulating immune cells such as monocytes, T cells and

  11. Expression of assayable residual stem cell damage in erythroid differentiation

    International Nuclear Information System (INIS)

    Huebner, G.E.; Miller, M.E.; Cronkite, E.P.

    1985-01-01

    In rodents, residual damage is inducible in hematopoietic stem cells by exposure to ionizing radiation or alkylating agents. This damage can b e assayed in mice by transferring bone marrow into lethally irradiated syngeneic recipients and subsequently measuring the incremental increase of-( 125 I)iodo-2'-deoxyuridine incorporation in spleens. In this study, bone marrow from mice treated 3 weeks previously with Methylnitrosourea (50 mg/kg) or 450 rad was injected into recipients in order to determine possible residual effects of treatment of erythroid cell differentiation following stem cell seeding. Such effects were detected by a reduced amount of 59 Fe incorporation into spleens, thus indicatin g transfer of residual stem cell damage to differentiating cells. (orig.)

  12. Microfluidic isolation of platelet-covered circulating tumor cells.

    Science.gov (United States)

    Jiang, Xiaocheng; Wong, Keith H K; Khankhel, Aimal H; Zeinali, Mahnaz; Reategui, Eduardo; Phillips, Matthew J; Luo, Xi; Aceto, Nicola; Fachin, Fabio; Hoang, Anh N; Kim, Wooseok; Jensen, Annie E; Sequist, Lecia V; Maheswaran, Shyamala; Haber, Daniel A; Stott, Shannon L; Toner, Mehmet

    2017-10-11

    The interplay between platelets and tumor cells is known to play important roles in metastasis by enhancing tumor cell survival, tumor-vascular interactions, and escape from immune surveillance. However, platelet-covered circulating tumor cells (CTC) are extremely difficult to isolate due to masking or downregulation of surface epitopes. Here we describe a microfluidic platform that takes advantage of the satellite platelets on the surface of these "stealth" CTCs as a ubiquitous surface marker for isolation. Compared to conventional CTC enrichment techniques which rely on known surface markers expressed by tumor cells, platelet-targeted isolation is generally applicable to CTCs of both epithelial and mesenchymal phenotypes. Our approach first depletes unbound, free platelets by means of hydrodynamic size-based sorting, followed by immunoaffinity-based capture of platelet-covered CTCs using a herringbone micromixing device. This method enabled the reliable isolation of CTCs from 66% of lung and 60% of breast cancer (both epithelial) patient samples, as well as in 83% of melanoma (mesenchymal) samples. Interestingly, we observed special populations of CTCs that were extensively covered by platelets, as well as CTC-leukocyte clusters. Because these cloaked CTCs often escape conventional positive and negative isolation mechanisms, further characterization of these cells may uncover important yet overlooked biological information in blood-borne metastasis and cancer immunology.

  13. Detection and Characterization of Circulating Tumour Cells in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Liangxuan Zhang

    2016-06-01

    Full Text Available Multiple myeloma (MM remains an incurable disease despite recent therapeutic improvements. The ability to detect and characterize MM circulating tumour cells (CTCs in peripheral blood provides an alternative to replace or augment invasive bone marrow (BM biopsies with a simple blood draw, providing real-time, clinically relevant information leading to improved disease manage‐ ment and therapy selection. Here we have developed and qualified an enrichment-free, cell-based immunofluores‐ cence MM CTC assay that utilizes an automated digital pathology algorithm to distinguish MM CTCs from white blood cells (WBCs on the basis of CD138 and CD45 expression levels, as well as a number of morphological parameters. These MM CTCs were further characterized for expression of phospho-ribosomal protein S6 (pS6 as a readout for PI3K/AKT pathway activation. Clinical feasi‐ bility of the assay was established by testing blood samples from a small cohort of patients, where we detected popu‐ lations of both CD138pos and CD138neg MM CTCs. In this study, we developed an immunofluorescent cell-based assay to detect and characterize CTCs in MM.

  14. Circulating tumor cells in small-cell lung cancer : a predictive and prognostic factor

    NARCIS (Netherlands)

    Hiltermann, T J N; Pore, M M; van den Berg, Anke; Timens, W; Boezen, H M; Liesker, J J W; Schouwink, J H; Wijnands, W J A; Kerner, G S M A; Kruyt, F A E; Tissing, H; Tibbe, A G J; Terstappen, L W M M; Groen, H J M

    2012-01-01

    BACKGROUND: Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to

  15. Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

    Science.gov (United States)

    Nejlund, Sarah; Smith, Julie; Kraan, Jaco; Stender, Henrik; Van, Mai N; Langkjer, Sven T; Nielsen, Mikkel T; Sölétormos, György; Hillig, Thore

    2016-08-01

    A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based

  16. Isolation and Molecular Characterization of Circulating Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Xi Luo

    2014-05-01

    Full Text Available Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.

  17. Eliminating Hairy Cell Leukemia Minimal Residual Disease

    Science.gov (United States)

    In this trial, patients with hairy cell leukemia who have disease-related symptoms that require treatment will be randomly assigned to receive cladribine with either concurrent rituximab or rituximab at least 6 months after completing cladribine therapy.

  18. Detection of circulating breast cancer cells using photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran

    According to the American Cancer Society, more than 200,000 new cases of breast cancer are expected to be diagnosed this year. Moreover, about 40,000 women died from breast cancer last year alone. As breast cancer progresses in an individual, it can transform from a localized state to a metastatic one with multiple tumors distributed through the body, not necessarily contained within the breast. Metastasis is the spread of cancer through the body by circulating tumor cells (CTCs) which can be found in the blood and lymph of the diagnosed patient. Diagnosis of a metastatic state by the discovery of a secondary tumor can often come too late and hence, significantly reduce the patient's chance of survival. There is a current need for a CTC detection method which would diagnose metastasis before the secondary tumor occurs or reaches a size resolvable by current imaging systems. Since earlier detection would improve prognosis, this study proposes a method of labeling of breast cancer cells for detection with a photoacoustic flow cytometry system as a model for CTC detection in human blood. Gold nanoparticles and fluorescent polystyrene nanoparticles are proposed as contrast agents for T47D, the breast cancer cell line of choice. The labeling, photoacoustic detection limit, and sensitivity are first characterized and then applied to a study to show detection from human blood.

  19. Carboxybetaine methacrylate oligomer modified nylon for circulating tumor cells capture.

    Science.gov (United States)

    Dong, Chaoqun; Wang, Huiyu; Zhang, Zhuo; Zhang, Tao; Liu, Baorui

    2014-10-15

    Circulating tumor cells (CTC) capture is one of the most effective approaches in diagnosis and treatment of cancers in the field of personalized cancer medicine. In our study, zwitterionic carboxybetaine methacrylate (CBMA) oligomers were grafted onto nylon via atomic transfer random polymerization (ATRP) which would serve as a novel material for the development of convenient CTC capture interventional medical devices. The chemical, physical and biological properties of pristine and modified nylon surfaces were assessed by Fourier transform infrared spectra, atomic force microscope, water contact angle measurements, X-ray photoelectron spectroscopy, protein adsorption, platelet adhesion, and plasma recalcification time (PRT) determinations, etc. The results, including the significant decrease of proteins adsorption and platelets adhesion, as well as prolonged PRTs demonstrated the extraordinary biocompatibility and blood compatibility of the modified surface. Furthermore, we showed that upon immobilization of anti-epithelial cell adhesion molecular (anti-EpCAM) antibody onto the CBMA moiety, the modified nylon surface can selectively capture EpCAM positive tumor cells from blood with high efficiency, indicating the potential of the modified nylon in the manufacture of convenient interventional CTC capture medical devices. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Daily variation in radiosensitivity of circulating blood cells and bone marrow cell density in mice

    International Nuclear Information System (INIS)

    Tabatabai, R.N.

    1984-01-01

    Mice on a 12/12 light/dark cycle were bled during a twenty-four hour period each week for eight weeks to establish daily values of circulating blood cells. No significant daily variation was found in total red blood cells, hematocrit, or percentage of reticulocytes. A significant (P < 0.001) daily variation was found in total white blood cells, with the minimum occurring at 8 PM and the maximum occurring during the daylight hours from 8 a.m. to 2 p.m. Mice were then exposed to 0 R, 20 R, 50 R, or 100 R of x-radiation to determine what dose significantly reduces the total white cell count in circulating blood. It was found that 100 R significantly (P < .05) reduces the total white cell count over a four week period post-exposure. To determine if circulating blood cells and bone marrow cells show a diurnal radiosensitivity, mice were exposed to 100 R or 200 R of x-radiation at noon or midnight. Hematocrits, reticulocyte and white blood cell counts, daily white blood cell rhythm, and bone marrow cell density indicate that these mice were more radiosensitive at night

  1. Comparison of circulating and intratumoral regulatory T cells in patients with renal cell carcinoma.

    Science.gov (United States)

    Asma, Gati; Amal, Gorrab; Raja, Marrakchi; Amine, Derouiche; Mohammed, Chebil; Amel, Ben Ammar Elgaaied

    2015-05-01

    The clear evidence that tumor-infiltrating lymphocytes (TIL) exists in the tumor microenvironment raises the question why renal cell carcinoma (RCC) progresses. Numerous studies support the implication of CD4(+)CD25(high) regulatory T (Treg) cells in RCC development. We aimed in this study to characterize the phenotype and function of circulating and intratumoral Treg cells of RCC patient in order to evaluate their implication in the inhibition of the local antitumor immune response. Our results demonstrate that the proportion of Treg in TIL was, in average, similar to that found in circulating CD4(+) T cells of patients or healthy donors. However, intratumoral Treg exhibit a marked different phenotype when compared with the autologous circulating Treg. A higher CD25 mean level, HLA-DR, Fas, and GITR, and a lower CD45RA expression were observed in intratumoral Treg, suggesting therefore that these cells are effector in the tumor microenvironment. Additionally, intratumoral Treg showed a higher inhibitory function on autologous CD4(+)CD25(-) T cells when compared with circulating Treg that may be explained by an overexpression of FoxP3 transcription factor. These findings suggest that intratumoral Treg could be major actors in the impairment of local antitumor immune response for RCC patients.

  2. Tendon repair augmented with a novel circulating stem cell population.

    Science.gov (United States)

    Daher, Robert J; Chahine, Nadeen O; Razzano, Pasquale; Patwa, Sohum A; Sgaglione, Nicholas J; Grande, Daniel A

    2011-01-01

    Tendon ruptures are common sports-related injuries that are often treated surgically by the use of sutures followed by immobilization. However, tendon repair by standard technique is associated with long healing time and often suboptimal repair. Methods to enhance tendon repair time as well as the quality of repair are currently unmet clinical needs. Our hypothesis is that the introduction of a unique stem cell population at the site of tendon transection would result in an improved rate and quality of repair. Achilles tendons of fifty-one Sprague-Dawley rats were transected and suture-repaired. In half of the rats, a biodegradable scaffold seeded with allogenic circulating stem cells was placed as an onlay to the defect site in addition to the suture repair. The other half was treated with suture alone to serve as the control group. Animals were randomized to a two-, four-, or six-week time group. At the time of necropsy, tendons were harvested and prepared for either biomechanical or histological analysis. Histological slides were evaluated in a blinded fashion with the use of a grading scale. By two weeks, the experimental group demonstrated a significant improvement in repair compared to controls with no failures. Average histological scores of 0.6 and 2.6 were observed for the experimental and control group respectively. The experimental group demonstrated complete bridging of the transection site with parallel collagen fiber arrangement. By four weeks, both groups showed a continuing trend of healing, with the scaffold group exceeding the histological quality of the tissue repaired with suture alone. Biomechanically, the experimental group had a decreasing cross-sectional area with time which was also associated with a significant increase in the ultimate tensile strength of the tendons, reaching 4.2MPa by six weeks. The experimental group also achieved a significantly higher elastic toughness by six weeks and saw an increase in the tensile modulus, reaching

  3. Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas.

    Science.gov (United States)

    Adams, Daniel L; Adams, Diane K; Stefansson, Steingrimur; Haudenschild, Christian; Martin, Stuart S; Charpentier, Monica; Chumsri, Saranya; Cristofanilli, Massimo; Tang, Cha-Mei; Alpaugh, R Katherine

    2016-05-04

    Enumeration of circulating tumor cells (CTCs) isolated from the peripheral blood of breast cancer patients holds promise as a clinically relevant, minimally invasive diagnostic test. However, CTC utility has been limited as a prognostic indicator of survival by the inability to stratify patients beyond general enumeration. In comparison, histological biopsy examinations remain the standard method for confirming malignancy and grading malignant cells, allowing for cancer identification and then assessing patient cohorts for prognostic and predictive value. Typically, CTC identification relies on immunofluorescent staining assessed as absent/present, which is somewhat subjective and limited in its ability to characterize these cells. In contrast, the physical features used in histological cytology comprise the gold standard method used to identify and preliminarily characterize the cancer cells. Here, we superimpose the methods, cytologically subtyping CTCs labeled with immunohistochemical fluorescence stains to improve their prognostic value in relation to survival. In this single-blind prospective pilot study, we tracked 36 patients with late-stage breast cancer over 24 months to compare overall survival between simple CTC enumeration and subtyping mitotic CTCs. A power analysis (1-β = 0. 9, α = 0.05) determined that a pilot size of 30 patients was sufficient to stratify this patient cohort; 36 in total were enrolled. Our results confirmed that CTC number is a prognostic indicator of patient survival, with a hazard ratio 5.2, p = 0.005 (95 % CI 1.6-16.5). However, by simply subtyping the same population based on CTCs in cytological mitosis, the hazard ratio increased dramatically to 11.1, p cancer, (2) mitotic CTCs may significantly correlate with shortened overall survival, and (3) larger and more defined patient cohort studies are clearly called for based on this initial pilot study.

  4. Endovascular treatment of unruptured intracranial aneurysms and circulating endothelial cells.

    Science.gov (United States)

    Vendrell, Jean-François; Cezar, Renaud; Kuster, Nils; Lobotesis, Kyriakos; Costalat, Vincent; Machi, Paolo; Bonafe, Alain; Vendrell, Jean-Pierre

    2013-04-01

    To evaluate the potential implication of circulating endothelial cells (CECs) in complications following endovascular treatment (EVT) of unruptured intracranial aneurysms. CECs characterized as CD146(+)/CD105(+)/CD45(-)/DAPI(+) were considered to originate from an altered endothelial cell layer of the vessel wall. In 15 patients, CECs were characterized and enumerated by the CellTracks(®) System in blood samples from: (1) femoral artery (FA), (2) internal carotid artery (ICA) before (ICA1) and after procedure (ICA2), and (3) a peripheral vein before (PV1) and after EVT (PV2). Ischemic brain events were assessed using diffusion weighted imaging (DWI-MRI) before and 24h after EVT. In ICA1, the median number of single CECs and clusters of 2-5 CECs were higher than in FA, ICA2, PV1 and PV2 samples (P5 cells, sometimes >50μm, were mainly observed in ICA1 and never in PV1, PV2 or PV samples from ten healthy subjects. This distribution of CECs suggested femoral and ICA injury by the devices used, leading to endothelium shearing and desquamation of CECs. All patients discharged on day two (NIHSS score=0), however silent ischemic brain lesions were observed in 9/15 (60%). EVT detaches single and clusters of CECs from wall arteries that may be implicated in silent ischemic brain lesions genesis. Enumeration of CECs associated with DWI-MRI might represent an interesting strategy for monitoring and optimizing endovascular devices, and further limit EVT-related complications. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  5. Dynamic Fluctuation of Circulating Tumor Cells during Cancer Progression

    Directory of Open Access Journals (Sweden)

    Mazen A. Juratli

    2014-01-01

    Full Text Available Circulating tumor cells (CTCs are a promising diagnostic and prognostic biomarker for metastatic tumors. We demonstrate that CTCs’ diagnostic value might be increased through real-time monitoring of CTC dynamics. Using preclinical animal models of breast cancer and melanoma and in vivo flow cytometry with photoacoustic and fluorescence detection schematics, we show that CTC count does not always correlate with the primary tumor size. Individual analysis elucidated many cases where the highest level of CTCs was detected before the primary tumor starts progressing. This phenomenon could be attributed to aggressive tumors developing from cancer stem cells. Furthermore, real-time continuous monitoring of CTCs reveals that they occur at highly variable rates in a detection point over a period of time (e.g., a range of 0–54 CTCs per 5 min. These same fluctuations in CTC numbers were observed in vivo in epithelial and non-epithelial metastatic tumors, in different stages of tumor progression, and in different vessels. These temporal CTC fluctuations can explain false negative results of a one-time snapshot test in humans. Indeed, we observed wide variations in the number of CTCs in subsequent blood samples taken from the same metastatic melanoma patient, with some samples being CTC-free. If these phenomena are confirmed in our ongoing in vivo clinical trials, this could support a personalized strategy of CTC monitoring for cancer patients.

  6. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects

    Directory of Open Access Journals (Sweden)

    Minev Boris

    2010-04-01

    Full Text Available Abstract The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  7. Barotropic Eulerian residual circulation in the Gulf of California due to the M{sub 2} tide and wind stress

    Energy Technology Data Exchange (ETDEWEB)

    Argote, M. L.; Lavin, M. F.; Amador, A. [Departamento de Oceanografia Fisica, CICESE, Ensenada, Baja California (Mexico)

    1998-07-01

    A vertically integrated, non-linear numerical model in finite differences is used to analyze two forcing mechanisms of the mean barotropic circulation in the Gulf of California: topographic rectification due to tidal currents (M{sub 2}) and wind stress. Under tidal forcing the nonlinearities of the momentum equations induce unorganized strong tidal induced residual currents (u{sub e} > 5 cm s{sup -}1) in the channels between the islands, and along-isobath anticyclonic circulation in the Northern Gulf, with speeds u{sub 3} < 2.5 cm s{sup -}1 over the edge of Delfin Basin. These numerical results are in agreement with analytical results, which indicate that the tidal-induced currents are mostly due to the advective terms, and that continuity and the Coriolis term (but regulated by bottom friction) are responsible for the along-isobath flow. The quadratic bottom friction plays a role in generating mean currents only in the very shallow area off the Colorado River Delta. The effect of wind stress was modeled by imposing upon the running M{sub 2} model a constant surface stress ( r = 0.016 Pa), from the NW for winter conditions and from the SE for summer conditions. The wind-induced circulation was obtained by averaging over a tidal cycle and then subtracting the tidal residuals. The two wind directions produce almost identical circulation patterns, but with opposite directions. For the NW wind stress, the main features of the predicted circulation are: (a) In the Northern Gulf an anticyclonic circulation pattern, with the strongest currents (up to {approx} 10 cm s-1) following the bathymetry of the rim of Delfin Basin, Wagner Basin and the mainland coast off Bahia Adair and Bahia San Jorge. There is also a southward flow along the peninsula coast, from the Colorado River to Bahia San Luis Gonzaga. (b) In the Southern Gulf, there is a strong flow ({approx} 10 to 15 cm s{sup -}1) to the SE over the continental shelf along the mainland coast. A somewhat less well

  8. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    Directory of Open Access Journals (Sweden)

    Mark R. Openshaw

    2016-02-01

    Full Text Available Gestational trophoblastic neoplasia (GTN represents a group of diseases characterized by production of human chorionic gonadotropin (hCG. Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA (from 9% to 53% of total cfDNA in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis.

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

    Science.gov (United States)

    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.

  10. Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

    OpenAIRE

    Morrow, C. J.; Trapani, F.; Metcalf, R. L.; Bertolini, G.; Hodgkinson, C. L.; Khandelwal, G.; Kelly, P.; Galvin, M.; Carter, L.; Simpson, K. L.; Williamson, S.; Wirth, C.; Simms, N.; Frankliln, L.; Frese, K. K.

    2016-01-01

    Background Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, ?liquid biopsies? such as circulating tumour cells (CTCs) are minimally invasive...

  11. Automated detection of residual cells after sex-mismatched stem-cell transplantation – evidence for presence of disease-marker negative residual cells

    Directory of Open Access Journals (Sweden)

    Johannes Tilman

    2009-05-01

    Full Text Available Abstract Background A new chimerism analysis based on automated interphase fluorescence in situ hybridization (FISH evaluation was established to detect residual cells after allogene sex-mismatched bone marrow or blood stem-cell transplantation. Cells of 58 patients were characterized as disease-associated due to presence of a bcr/abl-gene-fusion or a trisomy 8 and/or a simultaneous hybridization of gonosome-specific centromeric probes. The automatic slide scanning platform Metafer with its module MetaCyte was used to analyse 3,000 cells per sample. Results Overall 454 assays of 58 patients were analyzed. 13 of 58 patients showed residual recipient cells at one stage of more than 4% and 12 of 58 showed residual recipient cells less than 4%, respectively. As to be expected, patients of the latter group were associated with a higher survival rate (48 vs. 34 month. In only two of seven patients with disease-marker positive residual cells between 0.1–1.3% a relapse was observed. Besides, disease-marker negative residual cells were found in two patients without relapse at a rate of 2.8% and 3.3%, respectively. Conclusion The definite origin and meaning of disease-marker negative residual cells is still unclear. Overall, with the presented automatic chimerism analysis of interphase FISH slides, a sensitive method for detection of disease-marker positive residual cells is on hand.

  12. Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

    International Nuclear Information System (INIS)

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1988-01-01

    Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells

  13. Detection of circulating tumour cells on mRNA levels with established breast cancer cell lines.

    Science.gov (United States)

    Zebisch, Michael; Kölbl, Alexandra C; Andergassen, Ulrich; Hutter, Stephan; Neugebauer, Julia; Engelstädter, Verena; Günthner-Biller, Maria; Jeschke, Udo; Friese, Klaus; Rack, Brigitte

    2013-03-01

    Circulating tumour cells were detected and quantified by real-time polymerase chain reaction (PCR) in peripheral blood, based on the fact that the expression of certain genes is upregulated in tumour tissues in comparison to surrounding blood cells. Calibration curves showing gene expression as functions of the number of tumour cells within a blood sample were prepared. Blood samples were therefore spiked with cells of breast cancer cell lines, RNA was extracted, transcribed to complementary DNA (cDNA) and used in real-time PCR reaction on the Cytokeratins (CK) 8, 18 and 19. Calibration curves were generated by Microsoft™ Excel®. Relative quantification curves of gene expression in different breast cancer cell lines showed no unitary tendencies. The oscillations in the relative quantification curves of gene expression suggested an occurrence of immunological effects, leading to an apparent agglutination of added tumour cells together with the blood cells of the sample. Thus, strategies to obtain evaluable results should be considered.

  14. Monitoring circulating gammadelta T cells in cancer patients to optimize gammadelta T cell-based immunotherapy

    OpenAIRE

    Hans-Heinrich eOberg; Christian eKellner; Matthias ePeipp; Susanne eSebens; Sabine eAdam-Klages; Martin eGramatzki; Dieter eKabelitz; Daniela eWesch

    2014-01-01

    The success of gammadelta (gd) T cell-based immunotherapy, where the cytotoxic activity of circulating gd T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients´ gd T cells. A possible influence of radio- or chemotherapy on gd T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily det...

  15. Gene expression of circulating tumour cells in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Bölke E

    2009-09-01

    Full Text Available Abstract Background The diagnostic tools to predict the prognosis in patients suffering from breast cancer (BC need further improvements. New technological achievements like the gene profiling of circulating tumour cells (CTC could help identify new prognostic markers in the clinical setting. Furthermore, gene expression patterns of CTC might provide important informations on the mechanisms of tumour cell metastasation. Materials and methods We performed realtime-PCR and multiplex-PCR analyses following immunomagnetic separation of CTC. Peripheral blood (PB samples of 63 patients with breast cancer of various stages were analyzed and compared to a control group of 14 healthy individuals. After reverse-transcription, we performed multiplex PCR using primers for the genes ga733.3, muc-1 and c-erbB2. Mammaglobin1, spdef and c-erbB2 were analyzed applying realtime-PCR. Results ga733.2 overexpression was found in 12.7% of breast cancer cases, muc-1 in 15.9%, mgb1 in 9.1% and spdef in 12.1%. In this study, c-erbB2 did not show any significant correlation to BC, possibly due to a highly ambient expression. Besides single gene analyses, gene profiles were additionally evaluated. Highly significant correlations to BC were found in single gene analyses of ga733.2 and muc-1 and in gene profile analyses of ga733.3*muc-1 and GA7 ga733.3*muc-1*mgb1*spdef. Conclusion Our study reveals that the single genes ga733.3, muc-1 and the gene profiles ga733.3*muc-1 and ga733.3*3muc-1*mgb1*spdef can serve as markers for the detection of CTC in BC. The multigene analyses found highly positive levels in BC patients. Our study indicates that not single gene analyses but subtle patterns of multiple genes lead to rising accuracy and low loss of specificity in detection of breast cancer cases.

  16. Evaluation of a multi-marker immunomagnetic enrichment assay for the quantification of circulating melanoma cells

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    Freeman James B

    2012-09-01

    Full Text Available Abstract Background Circulating melanoma cells (CMCs are thought to be valuable in improving measures of prognosis in melanoma patients and may be a useful marker of residual disease to identify non-metastatic patients requiring adjuvant therapy. We investigated whether immunomagnetic enrichment targeting multiple markers allows more efficient enrichment of CMCs from patient peripheral blood than targeting a single marker. Furthermore, we aimed to determine whether the number of CMCs in patient blood was associated with disease stage. Methods We captured CMCs by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271, both individually and in combination, by immunomagnetic enrichment. CMCs were enriched and quantified from the peripheral blood of 10 non-metastatic and 13 metastatic melanoma patients. Results Targeting all markers in combination resulted in the enrichment of more CMCs than when any individual marker was targeted (p  Conclusions Our results demonstrated that a combination of markers should be targeted for optimal isolation of CMCs. In addition, there are significantly more CMCs in metastatic patients compared with non-metastatic patients and therefore quantification of CMCs may prove to be a useful marker of disease progression.

  17. Fingolimod induces BAFF and expands circulating transitional B cells without activating memory B cells and plasma cells in multiple sclerosis.

    Science.gov (United States)

    Miyazaki, Yusei; Niino, Masaaki; Takahashi, Eri; Suzuki, Masako; Mizuno, Masanori; Hisahara, Shin; Fukazawa, Toshiyuki; Amino, Itaru; Nakano, Fumihito; Nakamura, Masakazu; Akimoto, Sachiko; Minami, Naoya; Fujiki, Naoto; Doi, Shizuki; Shimohama, Shun; Terayama, Yasuo; Kikuchi, Seiji

    2018-02-01

    Patients with multiple sclerosis (MS) who are treated with fingolimod have an increased proportion of transitional B cells in the circulation, but the underlying mechanism is not known. We hypothesized that B cell-activating factor of the tumor necrosis factor family (BAFF) is involved in the process. Compared with healthy controls and untreated MS patients, fingolimod-treated MS patients had significantly higher serum concentrations of BAFF, which positively correlated with the proportions and the absolute numbers of transitional B cells in blood. Despite the elevated concentrations of BAFF in fingolimod-treated MS patients, serum levels of soluble transmembrane activator and calcium-modulating cyclophilin ligand interactor, and B cell maturation antigen were not elevated. Our results show that fingolimod induces BAFF in the circulation and expands transitional B cells, but does not activate memory B cells or plasma cells in MS, which is favorable for the treatment of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Measurement of circulating cell-derived microparticles by flow cytometry: Sources of variability within the assay

    NARCIS (Netherlands)

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A.; Ferry, Berne

    2011-01-01

    Introduction: 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

  19. Circulating MiRNA-21 and programed cell death (PDCD) 4 gene ...

    African Journals Online (AJOL)

    Circulating MiRNA-21 and programed cell death (PDCD) 4 gene expression in hepatocellular carcinoma (HCC) in Egyptian patients. Gamalat El Gedawy, Manar Obada, Ayman Kelani, Hala El-Said, Naglaa M. Ghanayem ...

  20. Circulating rotavirus-specific T cells have a poor functional profile

    International Nuclear Information System (INIS)

    Parra, Miguel; Herrera, Daniel; Jácome, María Fernanda; Mesa, Martha C.; Rodríguez, Luz-Stella; Guzmán, Carolina; Angel, Juana; Franco, Manuel A.

    2014-01-01

    Frequencies of circulating T cells producing IFN-γ, TNF-α, and IL-2, and percentages of T cells proliferating after stimulation with rotavirus (RV), tetanus toxoid, and influenza were evaluated in PBMC derived from healthy adults and children. In addition, the potential anergic state of RV-specific T cells was analyzed by stimulation of PBMC with RV antigen in the presence of three anergy inhibitors (rIL-2, rIL-12, or DGKα-i). The quality and magnitude of RV-T cell responses were significantly lower than those of tetanus toxoid and influenza antigens. RV-CD4 T cell response was enriched in monofunctional IFN-γ + cells, while influenza-CD4 and tetanus toxoid-CD4 T cell responses were enriched in multifunctional T cells. Moreover, rIL-2 – unlike rIL-12 or DGKα-i – increased the frequencies of RV-CD4 TNF-α + , CD4 IFN-γ + , and CD8 IFN-γ + cells. Thus, circulating RV-T cells seem to have a relatively poor functional profile that may be partially reversed in vitro by the addition of rIL-2. - Highlights: • The quality and magnitude of circulating RV-T cell responses are relatively poor. • Circulating RV-CD4 T cells are enriched in monofunctional IFN-γ+ cells. • Treatment with rIL-2 increased the frequencies of cytokine secreting RV-T cells

  1. The shifted balance between circulating follicular regulatory T cells and follicular helper T cells in patients with ulcerative colitis.

    Science.gov (United States)

    Wang, Xinrui; Zhu, Yonggang; Zhang, Manli; Hou, Jie; Wang, Hongjuan; Jiang, Yanfang; Wang, Haifeng; Gao, Pujun

    2017-12-15

    B-cell immunity participates in the pathogenesis of ulcerative colitis (UC). The immune balance between follicular regulatory T (TFR) cells and follicular helper T (TFH) cells is important in regulating B-cell responses. However, the alteration of TFR/TFH balance in UC remains unclear. Peripheral blood from 25 UC patients and 15 healthy controls was examined for the frequencies of circulating TFR, TFH, and regulatory T (Treg) cells by flow cytometry. Levels of serum cytokines were measured using cytometric bead array (CBA). Disease activity was evaluated by the Mayo Clinic Score. Compared with controls, UC patients exhibited significant reductions in circulating Foxp3 + CXCR5 + TFR cells, the subset interleukin (IL)-10 + Foxp3 + CXCR5 + cells, and Treg cells, but significant expansions in Foxp3 - CXCR5 + TFH cells and IL-21 + Foxp3 - CXCR5 + cells. UC patients also had reduced levels of serum IL-10 and elevated levels of serum IL-21. The values of Mayo Clinic Score, C-reactive protein (CRP), or erythrocyte sedimentation rate (ESR) in UC patients were negatively correlated with circulating TFR cells, serum IL-10 level, and TFR/TFH ratio, while positively correlated with circulating TFH cells and serum IL-21 level. Alterations in circulating TFR and TFH cells shift the balance from immune tolerance to immune responsive state, contributing to dysregulated B-cell immunity and the pathogenesis of UC. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  2. Mutational analysis of circulating tumor cells from colorectal cancer patients and correlation with primary tumor tissue.

    Directory of Open Access Journals (Sweden)

    Anna Lyberopoulou

    Full Text Available Circulating tumor cells (CTCs provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3 mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

  3. Cornering metastases: therapeutic targeting of circulating tumor cells and stem cells.

    Directory of Open Access Journals (Sweden)

    Bishoy eFaltas

    2012-07-01

    Full Text Available The last decade has witnessed an evolution of our understanding of the biology of the metastatic cascade. Recent insights into the metastatic process show that it is complex, dynamic and multi-directional. This process starts at a very early stage in the natural history of solid tumor growth leading to early development of metastases that grow in parallel with the primary tumor. The role of stem cells in perpetuating cancer metastases is increasingly becoming more evident. At the same time, there is a growing recognition of the crucial role circulating tumor cells (CTCs play in the development of metastases. These insights have laid the biological foundations for therapeutic targeting of CTCs, a promising area of research that aims to reduce cancer morbidity and mortality by preventing the development of metastases at a very early stage. The hematogenous transport phase of the metastatic cascade provides critical access to CTCs for therapeutic targeting aiming to interrupt the metastatic process. Recent advances in the fields of nanotechnology and micro-fluidics have led to the development of several devices for in-vivo targeting of CTC during transit in the circulation. Selectin-coated tubes that target cell adhesion molecules, immuno-magnetic separators and in-vivo photoacoustic flow cytometers are currently being developed for this purpose. On the pharmacological front, several pharmacological and immunological agents targeting cancer stem cells are currently being developed. Such agents may ultimately prove to be effective against circulating tumor stem cells (CTSCs. Although still in its infancy, therapeutic targeting of CTCs and CTSCs offers an unprecedented opportunity to prevent the development of metastasis and potentially alter the natural history of cancer. By rendering cancer a local disease, these approaches could lead to major reductions in metastasis-related morbidity and mortality.

  4. Liquid Fluoride Salt Experimentation Using a Small Natural Circulation Cell

    Energy Technology Data Exchange (ETDEWEB)

    Yoder Jr, Graydon L [ORNL; Heatherly, Dennis Wayne [ORNL; Williams, David F [ORNL; Elkassabgi, Yousri M. [Texas A& M University, Kingsville; Caja, Joseph [Electrochemical Systems, Inc.; Caja, Mario [ORNL; Jordan, John [Texas A& M University, Kingsville; Salinas, Roberto [Texas A& M University, Kingsville

    2014-04-01

    A small molten fluoride salt experiment has been constructed and tested to develop experimental techniques for application in liquid fluoride salt systems. There were five major objectives in developing this test apparatus: Allow visual observation of the salt during testing (how can lighting be introduced, how can pictures be taken, what can be seen) Determine if IR photography can be used to examine components submerged in the salt Determine if the experimental configuration provides salt velocity sufficient for collection of corrosion data for future experimentation Determine if a laser Doppler velocimeter can be used to quantify salt velocities. Acquire natural circulation heat transfer data in fluoride salt at temperatures up to 700oC All of these objectives were successfully achieved during testing with the exception of the fourth: acquiring velocity data using the laser Doppler velocimeter. This paper describes the experiment and experimental techniques used, and presents data taken during natural circulation testing.

  5. Red blood cell damage from extracorporeal circulation in hemodialysis.

    Science.gov (United States)

    Polaschegg, Hans-Dietrich

    2009-01-01

    Blood damage is an unavoidable side effect of extracorporeal circulation. The effects of blood damage on patients' hematocrit and erythropoietin requirement as well as other potential side effects have not been studied for uneventful treatments. Comparing long nocturnal dialysis with regular 4-hour, three times per week dialysis allows for the conclusion that the influence of blood damage caused by extracorporeal circulation is small compared with biochemical effects. Acute hemolysis is one of the few remaining mechanical problems of dialysis. Acute hemolysis is caused by obstructions within the extracorporeal circuit caused by manufacturing errors, kinking of blood tubing or user errors, or by a combination of excessive flow and improper cannula or catheter dimensions. The risk of acute hemolysis can be further reduced by industrial quality control, better design of dialysis equipment, and hemodialysis machine control. Adverse effects caused by chronic mechanical hemolysis need to be studied.

  6. Morphological differences between circulating tumor cells from prostate cancer patients and cultured prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Sunyoung Park

    Full Text Available Circulating tumor cell (CTC enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

  7. Detection of circulating tumor cells in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Annkathrin eHanssen

    2015-09-01

    Full Text Available Lung Cancer is the most common cause of cancer related deaths that frequently metastasizes prior to disease diagnosis. Circulating tumor cells (CTCs are found in many different types of epithelial tumors and are of great clinical interest in terms of prognosis and therapy intervention. Here, we present and discuss EpCAM-dependent and -independent capture of CTCs in non-small cell lung cancer (NSCLC and the clinical relevance of CTC detection and characterization. Taking blood samples and analyzing CTCs as liquid biopsy might be a far less invasive diagnostic strategy than biopsies of lung tumors or metastases. Moreover, sequential blood sampling allows to study the dynamic changes of tumor cells during therapy, in particular the development of resistant tumor cell clones.

  8. Multivalent binding and biomimetic cell rolling improves the sensitivity and specificity of circulating tumor cell capture.

    Science.gov (United States)

    Myung, Ja Hye; Eblan, Michael J; Caster, Joseph M; Park, Sinjung; Poellmann, Michael J; Wang, Kyle; Tepper, Joel E; Tam, Kevin A; Miller, Seth M; Shen, Colette; Chen, Ronald C; Zhang, Tian; Chera, Bhishamjit; Wang, Andrew Z; Hong, Seungpyo

    2018-03-15

    We aimed to examine the effects of multivalent binding and biomimetic cell rolling on the sensitivity and specificity of circulating tumor cell (CTC) capture. We also investigated the clinical significance of CTCs and their kinetic profiles in cancer patients undergoing radiotherapy (RT) treatment. Patients with histologically confirmed primary carcinoma undergoing RT, with or without chemotherapy, were eligible for enrollment. Peripheral blood was collected prospectively at up to 5 time points, including prior to RT, at the first week, mid-point and final week of treatment, as well as 4 to 12 weeks after completion of RT. CTC capture was accomplished using a nanotechnology-based assay (CapioCyte) functionalized with aEpCAM, aHER-2, and aEGFR. CapioCyte was able to detect CTCs in all 24 cancer patients enrolled. Multivalent binding via poly(amidoamine) dendrimers further improved capture sensitivity. We also showed that cell rolling effect can improve CTC capture specificity (% of captured cells that are CK+/CD45-/DAPI+) up to 38%. Among the 18 patients with sequential CTC measurements, the median CTC decreased from 113 CTCs/mL before RT to 32 CTCs/mL at completion of RT (p = 0.001). CTCs declined throughout RT in patients with complete clinical and/or radiographic response, in contrast to an elevation in CTCs at mid or post-RT in the 2 patients with known pathologic residual disease. Our study demonstrated that multivalent binding and cell rolling can improve the sensitivity and specificity of CTC capture compared to multivalent binding alone, allowing reliable monitoring of CTC changes during and after treatment. Copyright ©2018, American Association for Cancer Research.

  9. Clinical significance of circulating blood and endothelial cell microparticles in sickle-cell disease.

    Science.gov (United States)

    Kasar, Mutlu; Boğa, Can; Yeral, Mahmut; Asma, Suheyl; Kozanoglu, Ilknur; Ozdogu, Hakan

    2014-01-01

    Increased thrombocyte activation leads to a higher likelihood of coagulation in sickle-cell disease. On the other hand, chronic inflammation and endothelial cell activation promote vaso-occlusion. The effect of circulating microparticles derived from erythrocytes, monocytes, thrombocytes, and endothelial cells on the vaso-occlusive process is unclear. This study aims to analyze the relationship between sickle-cell disease and miscellaneous organ complications by defining the circulating microparticles during the steady-state and painful crisis periods in 45 patients with sickle-cell disease. Microparticle analysis was conducted using an eight-parameter flow cytometric method, using CD61 PERCP, CD142PE, CD106 FITC, CD14 APC-H7, CD235a FITC, and Annexin-V APC monoclonal antibodies. Microparticle levels of sickle-cell patients were found to be significantly higher during both painful crisis and steady-state situations compared with the control group (for all, p microparticles, levels of erythrocyte microparticles (eMPs) were significantly higher during crisis than in the steady-state period (eMP steady state vs. painful crisis: 7.59 ± 12.24 vs. 7.59 ± 12.24, respectively; p Microparticles, including eMPs, were not affected by hydroxyurea treatment. Their level did not reflect the high frequency of crisis (>3 times/year). Thrombocyte microparticle levels were found to be higher in patients with nephropathia than in those without (48.05 ± 40.23 vs. 7.67 ± 6.75, respectively; p microparticles seem to be involved in the pathogenesis of sickle-cell disease. eMPs may help with the management of crisis. Thrombocyte microparticles might predict renal damage induced by vaso-occlusion.

  10. The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells

    Directory of Open Access Journals (Sweden)

    Williams Michael J

    2009-03-01

    Full Text Available Abstract Background When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Results Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1 fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. Conclusion The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At

  11. Molecular characterization of circulating plasma cells in patients with active systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Patricia L Lugar

    Full Text Available Systemic lupus erythematosus (SLE is a generalized autoimmune disease characterized by abnormal B cell activation and the occurrence of increased frequencies of circulating plasma cells (PC. The molecular characteristics and nature of circulating PC and B cells in SLE have not been completely characterized. Microarray analysis of gene expression was used to characterize circulating PC in subjects with active SLE. Flow cytometry was used to sort PC and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations were then compared. SLE PC exhibited a similar gene expression signature as tonsil PC. The differences in gene expression between SLE PC and normal tonsil PC and tonsil plasmablasts (PB suggest a mature Ig secreting cell phenotype in the former population. Despite this, SLE PC differed in expression of about half the genes from previously published gene expression profiles of normal bone marrow PC, indicating that these cells had not achieved a fully mature status. Abnormal expression of several genes, including CXCR4 and S1P(1, suggests a mechanism for the persistence of SLE PC in the circulation. All SLE B cell populations revealed an interferon (IFN gene signature previously only reported in unseparated SLE peripheral blood mononuclear cells. These data indicate that SLE PC are a unique population of Ig secreting cells with a gene expression profile indicative of a mature, but not fully differentiated phenotype.

  12. Circulating dendritic cells in pediatric patients with nephrotic syndrome

    African Journals Online (AJOL)

    EL-HAKIM

    immune system in the pathogenesis of idiopathic. NS3. DCs are rare, ubiquitously distributed, migratory antigen presenting cells (APCs), derived from. CD34 bone marrow stem cells. In addition to having the unique capacity to prime naive T cells,. DCs also regulate various effector cell functions and play central roles in ...

  13. Identification of circulating fetal cell markers by microarray analysis

    DEFF Research Database (Denmark)

    Brinch, Marie; Hatt, Lotte; Singh, Ripudaman

    2012-01-01

    OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found...... identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem...

  14. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  15. Identification of critical residues of linear B cell epitope on Goodpasture autoantigen.

    Directory of Open Access Journals (Sweden)

    Xiao-yu Jia

    Full Text Available The autoantigen of anti-glomerular basement membrane (GBM disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IVNC1. Our previous study revealed a peptide on α3(IVNC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150. This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA.We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001. P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001 of antibody reaction after each residue replacement.We defined GFxF (α3142, 143,145 as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.

  16. The rationale for liquid biopsy in colorectal cancer: a focus on circulating tumor cells.

    Science.gov (United States)

    Gazzaniga, Paola; Raimondi, Cristina; Nicolazzo, Chiara; Carletti, Raffaella; di Gioia, Cira; Gradilone, Angela; Cortesi, Enrico

    2015-01-01

    Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells.

  17. Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

    Science.gov (United States)

    Fachin, Fabio; Spuhler, Philipp; Martel-Foley, Joseph M; Edd, Jon F; Barber, Thomas A; Walsh, John; Karabacak, Murat; Pai, Vincent; Yu, Melissa; Smith, Kyle; Hwang, Henry; Yang, Jennifer; Shah, Sahil; Yarmush, Ruby; Sequist, Lecia V; Stott, Shannon L; Maheswaran, Shyamala; Haber, Daniel A; Kapur, Ravi; Toner, Mehmet

    2017-09-07

    Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm-50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15-20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

  18. Residual stresses in a co-sintered SOC half-cell during post-sintering cooling

    DEFF Research Database (Denmark)

    Charlas, Benoit; Chatzichristodoulou, Christodoulos; Brodersen, Karen

    2014-01-01

    Due to the thermal expansion mismatch between the layers of a Solid Oxide Cell, residual stresses (thermal stresses) develop during the cooling after sintering. Residual stresses can induce cell curvature for asymmetric cells but more importantly they also result in more fragile cells. Depending...

  19. Strenuous exercise decreases the percentage of type 1 T cells in the circulation

    DEFF Research Database (Denmark)

    Steensberg, A; Toft, A D; Bruunsgaard, H

    2001-01-01

    Prolonged strenuous exercise is followed by a temporary functional immune impairment. Low numbers of CD4+ T helper (Th) and CD8+ T cytotoxic (Tc) cells are found in the circulation. These cells can be divided according to their cytokine profile into type 1 (Th1 and Tc1), which produce interferon...

  20. The Emerging Role of Circulating Tumor Cell Detection in Genitourinary Cancer

    NARCIS (Netherlands)

    Small, A.C.; Gong, Y.X.; Oh, W.K.; Hall, S.J.; Rijn, van C.J.M.; Galsky, M.D.

    2012-01-01

    Purpose: Circulating tumor cells are malignant cells in peripheral blood that originate from primary tumors or metastatic sites. The heterogeneous natural history and propensity for recurrence in prostate, bladder and kidney cancers are well suited for improved individualization of care using

  1. A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

    Science.gov (United States)

    Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

    2016-05-06

    Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after

  2. Characterization of circulating natural killer cells in neotropical primates.

    Directory of Open Access Journals (Sweden)

    Angela Carville

    Full Text Available Despite extensive use of nonhuman primates as models for infectious diseases and reproductive biology, imprecise phenotypic and functional definitions exist for natural killer (NK cells. This deficit is particularly significant in the burgeoning use of small, less expensive New World primate species. Using polychromatic flow cytometry, we identified peripheral blood NK cells as CD3-negative and expressing a cluster of cell surface molecules characteristic of NK cells (i.e., NKG2A, NKp46, NKp30 in three New World primate species - common marmosets, cotton-top tamarins, and squirrel monkeys. We then assessed subset distribution using the classical NK markers, CD56 and CD16. In all species, similar to Old World primates, only a minor subset of NK cells was CD56+, and the dominant subset was CD56-CD16+. Interestingly, CD56+ NK cells were primarily cytokine-secreting cells, whereas CD56-CD16+ NK cells expressed significantly greater levels of intracellular perforin, suggesting these cells might have greater potential for cytotoxicity. New World primate species, like Old World primates, also had a minor CD56-CD16- NK cell subset that has no obvious counterpart in humans. Herein we present phenotypic profiles of New World primate NK cell subpopulations that are generally analogous to those found in humans. This conservation among species should support the further use of these species for biomedical research.

  3. Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells

    NARCIS (Netherlands)

    Gijsbers, Linda; Koel, Björn; Weggeman, Miranda; Goudsmit, Jaap; Havenga, Menzo; Marzio, Giuseppe

    2005-01-01

    Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for

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

    Science.gov (United States)

    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 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.

  5. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    David T. Ting

    2014-09-01

    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

  6. Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

    Science.gov (United States)

    Ting, David T; Wittner, Ben S; Ligorio, Matteo; Vincent Jordan, Nicole; Shah, Ajay M; Miyamoto, David T; Aceto, Nicola; Bersani, Francesca; Brannigan, Brian W; Xega, Kristina; Ciciliano, Jordan C; Zhu, Huili; MacKenzie, Olivia C; Trautwein, Julie; Arora, Kshitij S; Shahid, Mohammad; Ellis, Haley L; Qu, Na; Bardeesy, Nabeel; Rivera, Miguel N; Deshpande, Vikram; Ferrone, Cristina R; Kapur, Ravi; Ramaswamy, Sridhar; Shioda, Toshi; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A

    2014-09-25

    Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Circulating tumor cells (Ctc) and kras mutant circulating free Dna (cfdna) detection in peripheral blood as biomarkers in patients diagnosed with exocrine pancreatic cancer

    International Nuclear Information System (INIS)

    Earl, Julie; Garcia-Nieto, Sandra; Martinez-Avila, Jose Carlos; Montans, José; Sanjuanbenito, Alfonso; Rodríguez-Garrote, Mercedes; Lisa, Eduardo; Mendía, Elena; Lobo, Eduardo; Malats, Núria; Carrato, Alfredo; Guillen-Ponce, Carmen

    2015-01-01

    Pancreatic cancer remains one of the most difficult cancers to treat with the poorest prognosis. The key to improving survival rates in this disease is early detection and monitoring of disseminated and residual disease. However, this is hindered due to lack reliable diagnostic and predictive markers which mean that the majority of patients succumb to their condition within a few months. We present a pilot study of the detection circulating free DNA (cfDNA) combined with tumor specific mutation detection by digital PCR as a novel minimally invasive biomarker in pancreatic ductal adenocarcinoma (PDAC). This was compared to the detection of CTC by the CellSearch® system and a novel CTC enrichment strategy based on CD45 positive cell depletion. The aim of the study was to assess tumor specific DNA detection in plasma and CTC detection as prognostic markers in PDAC. We detected KRAS mutant cfDNA in 26 % of patients of all stages and this correlated strongly with Overall Survival (OS), 60 days (95 % CI: 19–317) for KRAS mutation positive vs 772 days for KRAS mutation negative (95 % CI: 416–1127). Although, the presence of CTC detected by the CellSearch® system did correlate significantly with OS, 88 days (95 % CI: 27–206) CTC positive vs 393 days CTC negative (95 % CI: 284–501), CTC were detected in only 20 % of patients, the majority of which had metastatic disease, whereas KRAS mutant cfDNA was detected in patients with both resectable and advanced disease. Tumor specific cfDNA detection and CTC detection are promising markers for the management of patients with PDAC, although there is a need to validate these results in a larger patient cohort and optimize the detection of CTC in PDAC by applying the appropriate markers for their detection. The online version of this article (doi:10.1186/s12885-015-1779-7) contains supplementary material, which is available to authorized users

  8. Circulating tumor cells and their relationship with clinical and morphological characteristics of colorectal cancer

    Directory of Open Access Journals (Sweden)

    O I Kit

    2018-02-01

    Full Text Available Aim. To investigate the dependence of the number of circulating tumor cells in peripheral blood of colorectal cancer patients on the clinical and morphological characteristics of underlying disease. Methods. 91 patients with verified metastatic colorectal cancer Т3-4N1-2М1 were included in the study. The average age of the patients was 61.5±1.7 years. The patients were divided into the study group (laparoscopic surgical treatment, n=44 and control group (open surgical intervention, n=47. The number of circulating tumor cells was determined in CellSearch™ system in the peripheral blood drawn before the intervention. The study of the association of attributes by constructing contingency tables consisted in calculating Pearson’s contingency coefficient c2 with Mantel-Haenszel correction for likelihood (nonparametric correction, estimating statistical significance of contingency and analyzing the tightness of the association by A. Chuprov’s mutual contingency coefficient. Results. We found contingency of the number of circulating tumor cells with clinical and morphological parameters of patients with colorectal cancer. The relationship between potential risk factors and increase of the number of circulating tumor cells in the peripheral blood was observed in all colorectal cancer patients, regardless of the surgical intervention method. The most pronounced association of the number of circulating tumor cells in the peripheral blood of metastatic colorectal cancer patients before surgery according to the mutual contingency coefficient (K was shown to be with present distant metastases (status M1b; K=0.63, p=0.0001 and stage T4 (K=0.56, p=0.0009. Conclusion. The obtained results emphasize the important predictive significance of the circulating tumor cells level in peripheral blood for assessment of the potential for colorectal cancer progression.

  9. Mobilization of Viable Tumor Cells Into the Circulation During Radiation Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Olga A. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); Anderson, Robin L. [The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); Metastasis Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Russell, Prudence A. [Department of Anatomical Pathology, St. Vincent Hospital, Fitzroy, VIC (Australia); Ashley Cox, R. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Ivashkevich, Alesia [Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Laboratory of DNA Repair and Genomics, Centre for Innate Immunity and Infectious Disease, Monash Institute for Medical Research, Monash University, Clayton, VIC (Australia); Swierczak, Agnieszka; Doherty, Judy P. [Metastasis Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Jacobs, Daphne H.M. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Smith, Jai [Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Siva, Shankar; Daly, Patricia E. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Ball, David L. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); and others

    2014-02-01

    Purpose: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. Methods and Materials: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. Results: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. Conclusions: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies.

  10. Date Palm (Phoenix dactylifera) Fruits as a Potential Cardioprotective Agent: The Role of Circulating Progenitor Cells

    Science.gov (United States)

    Alhaider, Ibrahim A.; Mohamed, Maged E.; Ahmed, K. K. M.; Kumar, Arun H. S.

    2017-01-01

    Context: Date palms, along with their fruits’ dietary consumption, possess enormous medicinal and pharmacological activities manifested in their usage in a variety of ailments in the various traditional systems of medicine. In recent years, the identification of progenitor cells in the adult organ systems has opened an altogether new approach to therapeutics, due to the ability of these cells to repair the damaged cells/tissues. Hence, the concept of developing therapeutics, which can mobilize endogenous progenitor cells, following tissue injury, to enhance tissue repair process is clinically relevant. Objectives: The present study investigates the potential of date of palm fruit extracts in repairing tissue injury following myocardial infarction (MI) potentially by mobilizing circulating progenitor cells. Methods: Extracts of four different varieties of date palm fruits common in Saudi Arabia eastern provision were scrutinized for their total flavonoid, total phenolic, in vitro antioxidant capacity, as well as their effects on two different rodent MI models. Results: High concentrations of phenolic and flavonoid compounds were observed in date palm fruit extracts, which contributed to the promising antioxidant activities of these extracts and the observed high protective effect against various induced in vivo MI. The extracts showed ability to build up reserves and to mobilize circulating progenitor cells from bone marrow and peripheral circulation to the site of myocardial infraction. Conclusion: Date palm fruit extracts have the potential to mobilize endogenous circulating progenitor cells, which can promote tissue repair following ischemic injury. PMID:28928656

  11. Date Palm (Phoenix dactylifera) Fruits as a Potential Cardioprotective Agent: The Role of Circulating Progenitor Cells.

    Science.gov (United States)

    Alhaider, Ibrahim A; Mohamed, Maged E; Ahmed, K K M; Kumar, Arun H S

    2017-01-01

    Context: Date palms, along with their fruits' dietary consumption, possess enormous medicinal and pharmacological activities manifested in their usage in a variety of ailments in the various traditional systems of medicine. In recent years, the identification of progenitor cells in the adult organ systems has opened an altogether new approach to therapeutics, due to the ability of these cells to repair the damaged cells/tissues. Hence, the concept of developing therapeutics, which can mobilize endogenous progenitor cells, following tissue injury, to enhance tissue repair process is clinically relevant. Objectives: The present study investigates the potential of date of palm fruit extracts in repairing tissue injury following myocardial infarction (MI) potentially by mobilizing circulating progenitor cells. Methods: Extracts of four different varieties of date palm fruits common in Saudi Arabia eastern provision were scrutinized for their total flavonoid, total phenolic, in vitro antioxidant capacity, as well as their effects on two different rodent MI models. Results: High concentrations of phenolic and flavonoid compounds were observed in date palm fruit extracts, which contributed to the promising antioxidant activities of these extracts and the observed high protective effect against various induced in vivo MI. The extracts showed ability to build up reserves and to mobilize circulating progenitor cells from bone marrow and peripheral circulation to the site of myocardial infraction. Conclusion: Date palm fruit extracts have the potential to mobilize endogenous circulating progenitor cells, which can promote tissue repair following ischemic injury.

  12. Mobilization of Viable Tumor Cells Into the Circulation During Radiation Therapy

    International Nuclear Information System (INIS)

    Martin, Olga A.; Anderson, Robin L.; Russell, Prudence A.; Ashley Cox, R.; Ivashkevich, Alesia; Swierczak, Agnieszka; Doherty, Judy P.; Jacobs, Daphne H.M.; Smith, Jai; Siva, Shankar; Daly, Patricia E.; Ball, David L.

    2014-01-01

    Purpose: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. Methods and Materials: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. Results: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. Conclusions: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies

  13. Circulating rotavirus-specific T cells have a poor functional profile

    Energy Technology Data Exchange (ETDEWEB)

    Parra, Miguel; Herrera, Daniel [Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Pontificia Universidad Javeriana, Carrera 7 # 40-62, Bogotá (Colombia); Jácome, María Fernanda; Mesa, Martha C. [Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá (Colombia); Rodríguez, Luz-Stella [Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Pontificia Universidad Javeriana, Carrera 7 # 40-62, Bogotá (Colombia); Guzmán, Carolina [Departamento de Pediatría, Hospital Universitario San Ignacio, Facultad de Medicina, Pontificia Universidad Javeriana, Bogotá (Colombia); Angel, Juana; Franco, Manuel A. [Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Pontificia Universidad Javeriana, Carrera 7 # 40-62, Bogotá (Colombia)

    2014-11-15

    Frequencies of circulating T cells producing IFN-γ, TNF-α, and IL-2, and percentages of T cells proliferating after stimulation with rotavirus (RV), tetanus toxoid, and influenza were evaluated in PBMC derived from healthy adults and children. In addition, the potential anergic state of RV-specific T cells was analyzed by stimulation of PBMC with RV antigen in the presence of three anergy inhibitors (rIL-2, rIL-12, or DGKα-i). The quality and magnitude of RV-T cell responses were significantly lower than those of tetanus toxoid and influenza antigens. RV-CD4 T cell response was enriched in monofunctional IFN-γ{sup +} cells, while influenza-CD4 and tetanus toxoid-CD4 T cell responses were enriched in multifunctional T cells. Moreover, rIL-2 – unlike rIL-12 or DGKα-i – increased the frequencies of RV-CD4 TNF-α{sup +}, CD4 IFN-γ{sup +}, and CD8 IFN-γ{sup +} cells. Thus, circulating RV-T cells seem to have a relatively poor functional profile that may be partially reversed in vitro by the addition of rIL-2. - Highlights: • The quality and magnitude of circulating RV-T cell responses are relatively poor. • Circulating RV-CD4 T cells are enriched in monofunctional IFN-γ+ cells. • Treatment with rIL-2 increased the frequencies of cytokine secreting RV-T cells.

  14. TUMOR-RELATED METHYLATED CELL-FREE DNA AND CIRCULATING TUMOR CELLS IN MELANOMA

    Directory of Open Access Journals (Sweden)

    Francesca eSalvianti

    2016-01-01

    Full Text Available Solid tumor release into the circulation cell-free DNA (cfDNA and circulating tumor cells (CTCs which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma.The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs.RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC.The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic than in healthy subjects (Pearson chi-squared test, p<0.001. The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive

  15. The association between circulating endothelial progenitor cells and coronary collateral formation.

    Science.gov (United States)

    Tokgözoğlu, Lale; Yorgun, Hikmet; Gürses, Kadri Murat; Canpolat, Uğur; Ateş, Ahmet Hakan; Tülümen, Erol; Kaya, Ergün Barış; Aytemir, Kudret; Kabakçı, Giray; Tuncer, Murat; Oto, Ali

    2011-12-01

    We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography. Circulating CD133(+)/34(+) and CD34(+)/KDR(+) EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133(+)/34(+) (p collateral formation (p collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037). In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Macrophages eliminate circulating tumor cells after monoclonal antibody therapy

    NARCIS (Netherlands)

    Gül, Nuray; Babes, Liane; Siegmund, Kerstin; Korthouwer, Rianne; Bögels, Marijn; Braster, Rens; Vidarsson, Gestur; ten Hagen, Timo L. M.; Kubes, Paul; van Egmond, Marjolein

    2014-01-01

    The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved.

  17. The effects of renal transplantation on circulating dendritic cells

    NARCIS (Netherlands)

    D.A. Hesselink (Dennis); L.M.B. Vaessen (Leonard); W.C.J. Hop (Wim); W. Schoordijk-Verschoor (Wenda); J.N.M. IJzermans (Jan); C.C. Baan (Carla); W. Weimar (Willem)

    2005-01-01

    textabstractThe effects of immunosuppressive agents on T cell function have been well characterized but virtually nothing is known about the effects of renal transplantation on human dendritic cells (DCs). With the use of flow cytometry, we studied the kinetics of myeloid and plasmacytoid DCs in

  18. Excess circulating alternatively activated myeloid (M2 cells accelerate ALS progression while inhibiting experimental autoimmune encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Ilan Vaknin

    Full Text Available Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs, representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease.We tested this working hypothesis in amyotrophic lateral sclerosis (ALS and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2 cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1 mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE, which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS, revealed a two-fold increase in the percentage of circulating MDSCs (LIN(-/LowHLA-DR(-CD33(+ compared to controls.Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high level of suppressive immune cells might

  19. A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells.

    Science.gov (United States)

    Takamatsu, H-H; Denyer, M S; Wileman, T E

    2002-09-10

    A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.

  20. Evaluation of Isolation Methods for Circulating Tumor Cells (CTCs

    Directory of Open Access Journals (Sweden)

    Galatea Kallergi

    2016-11-01

    Full Text Available Background: Detection of CTCs is a poor prognostic factor for many cancer types; however, their very low frequency represents an obstacle for their detection. The objective of the current study was to compare the performance of commonly used methods for CTCs isolation. Methods: The evaluated methods using spiking experiments of MCF7, SKBR3 and MDA MB-231 breast cancer cell lines were (i ficoll density gradient separation (DGS, (ii red blood cell lysis (Erythrolysis isolation, (iii positive immunomagnetic selection (EpCAM Dynal beads, (iv two different negative immunomagnetic separation systems (Dynal vs Miltenyi CD45 beads as well as (v the Cell Search platform and (vi the ISET system. Results: The recovery rates of Erythrolysis and DGS were 39% and 24%, respectively. Magnetic isolations are ranked from the worse to the best recovery rate as follows:, Myltenyi-anti-CD45 microbeads (24%; Dynal-anti-EpCAM beads (75%; Dynabeads-anti-CD45 (97%. CTCs isolation from blood samples using the CellSearch and ISET systems revealed that the recovery rate for Cell Search and ISET was 52% and 95%, respectively. Conclusions: Dynal-anti-CD45 beads have the best recovery rate compared to other magnetic methods. Furthermore the recovery rate of ISET was higher compared to Cell Search, especially for the more aggressive MDA-MB 231 cell line.

  1. Circulating endothelial cells and procoagulant microparticles in patients with glioblastoma: prognostic value.

    Directory of Open Access Journals (Sweden)

    Gaspar Reynés

    Full Text Available AIM: Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter. METHODS: Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification. RESULTS: Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001. After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99(th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004. CONCLUSION: Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.

  2. High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System

    OpenAIRE

    Regmi, Sagar; Fu, Afu; Luo, Kathy Qian

    2017-01-01

    Circulating tumor cells (CTCs) are the primary targets of cancer treatment as they cause distal metastasis. However, how CTCs response to exercise-induced high shear stress is largely unknown. To study the effects of hemodynamic microenvironment on CTCs, we designed a microfluidic circulatory system that produces exercise relevant shear stresses. We explore the effects of shear stresses on breast cancer cells with different metastatic abilities, cancer cells of ovarian, lung and leukemic orig...

  3. CCR 20th Anniversary Commentary: Paving the Way for Circulating Tumor Cells.

    Science.gov (United States)

    Allard, W Jeffrey; Terstappen, Leon W M M

    2015-07-01

    Cancer cells can enter the bloodstream, form distant metastases, and ultimately lead to death. A study by Allard and colleagues, which was published in the October 15, 2004, issue of Clinical Cancer Research, concluded that the CellSearch system could be used as a reliable tool to investigate circulating tumor cells and their clinical utility, and it spurred a still-growing interest in the field. ©2015 American Association for Cancer Research.

  4. Association between circulating osteoblast progenitor cells and aortic calcifications in women with postmenopausal osteoporosis.

    Science.gov (United States)

    Pirro, M; Manfredelli, M R; Schillaci, G; Helou, R S; Bagaglia, F; Melis, F; Scalera, G B; Scarponi, A M; Gentile, E; Mannarino, E

    2013-05-01

    Ectopic artery calcification has been documented in women with postmenopausal osteoporosis, in whom an imbalance in the number of circulating osteoprogenitor cells (OPCs) has been identified. Circulating OPCs form calcified nodules in vitro; however, it remains unknown whether an association exists between the number of circulating OPCs and aortic calcifications. We investigated the relationship between OPCs and aortic calcifications in women with postmenopausal osteoporosis. The number of circulating OPCs was quantified by FACS analysis in 50 osteoporotic postmenopausal women. OPCs were defined as CD15-/alkaline-phosphatase(AP)+ cells coexpressing or not CD34. Participants underwent measurement of markers of bone metabolism, bone mineral density and abdominal aortic calcium (AAC) by 64-slice computed tomography. Patients with AAC were older, had lower 25(OH)vitamin D levels and higher circulating CD15-/AP+/CD34- cells than those without AAC. Significant correlates of AAC included age (rho = 0.38 p = 0.006), calcium (rho = 0.35 p = 0.01), 25(OH)vitamin D (rho = -0.31, p = 0.03) and the number of CD15-/AP+/CD34- cells (rho = 0.55 p osteoporosis, the number of circulating CD15-/AP+/CD34- cells is significantly associated with increased aortic calcifications, that appear to be correlated also with reduced 25(OH)vitamin D levels. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Circulating tumor cells: clinically relevant molecular access based on a novel CTC flow cell.

    Directory of Open Access Journals (Sweden)

    Jessamine P Winer-Jones

    Full Text Available BACKGROUND: Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available. METHODOLOGY/PRINCIPAL FINDINGS: Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error and a precision (CV<25% down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL. CONCLUSIONS/SIGNIFICANCE: The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens

  6. Obstructive Apneas Induce Early Release of Mesenchymal Stem Cells into Circulating Blood

    Science.gov (United States)

    Carreras, Alba; Almendros, Isaac; Acerbi, Irene; Montserrat, Josep M.; Navajas, Daniel; Farré, Ramon

    2009-01-01

    Study Objectives: To investigate whether noninvasive application of recurrent airway obstructions induces early release of mesenchymal stem cells into the circulating blood in a rat model of obstructive sleep apnea. Design: Prospective controlled animal study. Setting: University laboratory. Patients or Participants: Twenty male Sprague-Dawley rats (250–300 g). Interventions: A specially designed nasal mask was applied to the anesthetized rats. Ten rats were subjected to a pattern of recurrent obstructive apneas (60 per hour, lasting 15 seconds each) for 5 hours. Ten anesthetized rats were used as controls. Measurements and Results: Mesenchymal stem cells from the blood and bone marrow samples were isolated and cultured to count the total number of colony-forming unit fibroblasts (CFU-F) of adherent cells after 9 days in culture. The number of CFU-F from circulating blood was significantly (P = 0.02) higher in the rats subjected to recurrent obstructive apneas (5.00 ± 1.16; mean ± SEM) than in controls (1.70 ± 0.72). No significant (P = 0.54) differences were observed in CFU-F from bone marrow. Conclusions: Application of a pattern of airway obstructions similar to those experienced by patients with sleep apnea induced an early mobilization of mesenchymal stem cells into circulating blood. Citation: Carreras A; Almendros I; Acerbi I; Montserrat JM; Navajas D; Farré R. Obstructive apneas induce early release of mesenchymal stem cells into circulating blood. SLEEP 2009;32(1):117-119. PMID:19189787

  7. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... ... High Sensitivity Bioanalyzer Chip (Agilent) to check for quality and size distribution. DNA library frag- ments were diluted, denatured and hybridized to a lawn of oligonucleotides immobilized on the flow-cell surface. Hy- bridized DNA template was amplified using immobilized oligonucleotides as primers.

  8. NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

    Science.gov (United States)

    Jan, Yu Jen; Chen, Jie-Fu; Zhu, Yazhen; Lu, Yi-Tsung; Chen, Szu Hao; Chung, Howard; Smalley, Matthew; Huang, Yen-Wen; Dong, Jiantong; Yu, Hsiao-Hua; Tomlinson, James S; Hou, Shuang; Agopian, Vatche G; Posadas, Edwin M; Tseng, Hsian-Rong

    2018-03-15

    Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC Assays

  9. Characterization of smooth muscle-like cells in circulating human peripheral blood.

    Science.gov (United States)

    Sugiyama, Seigo; Kugiyama, Kiyotaka; Nakamura, Shinichi; Kataoka, Keiichiro; Aikawa, Masanori; Shimizu, Koichi; Koide, Shunichi; Mitchell, Richard N; Ogawa, Hisao; Libby, Peter

    2006-08-01

    Smooth muscle cells play an important role in human vascular diseases. Several lines of evidence demonstrate that circulating smooth muscle precursor cells contribute to intimal hyperplasia in animal models. We obtained large spindle cells expressing alpha-smooth muscle actin (alpha-SMA), denoted here as "smooth muscle-like cells" (SMLC), from human peripheral blood mononuclear cells (PBMC). SMLC derived from human PBMC proliferated readily and expressed pro-inflammatory genes during early culture. After long-term culture, SMLC could contract and express characteristic smooth muscle cell markers. We found peripheral blood mononuclear cell expressing alpha-smooth muscle actin in the circulating blood that bore CD14 and CD105. Sorted CD14/CD105 double-positive PBMC could differentiate into SMLC. The number of CD14-CD105-bearing PBMC increased significantly in patients with coronary artery disease compared to patients without coronary artery disease. These results support the novel concept that smooth muscle precursor cells exist in circulating human blood and may contribute to the pathogenesis of vascular diseases.

  10. Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

    Science.gov (United States)

    DeJarnett, Natasha; Yeager, Ray; Conklin, Daniel J; Lee, Jongmin; O'Toole, Timothy E; McCracken, James; Abplanalp, Wes; Srivastava, Sanjay; Riggs, Daniel W; Hamzeh, Ihab; Wagner, Stephen; Chugh, Atul; DeFilippis, Andrew; Ciszewski, Tiffany; Wyatt, Brad; Becher, Carrie; Higdon, Deirdre; Ramos, Kenneth S; Tollerud, David J; Myers, John A; Rai, Shesh N; Shah, Jasmit; Zafar, Nagma; Krishnasamy, Sathya S; Prabhu, Sumanth D; Bhatnagar, Aruni

    2015-11-01

    Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways. © 2015 American Heart Association, Inc.

  11. Hydrogen peroxide inhibits exercise-induced increase of circulating stem cells with endothelial progenitor capacity.

    Science.gov (United States)

    Suvorava, Tatsiana; Kumpf, Stephanie; Rauch, Bernhard H; Dao, Vu Thao-Vi; Adams, Volker; Kojda, Georg

    2010-02-01

    The number of circulating stem cells with endothelial progenitor capacity (EPCs) inversely correlates with the number of cardiovascular risk factors. In this study we sought to investigate the effects of vascular H(2)O(2) on circulating EPC levels. In C57BL/6 mice 3 weeks of freely moving or forced physical activity or voluntary exercise failed to increase circulating EPCs defined as double positive for Flk-1 and CD34, CD133 or Sca-1. Likewise, neither insertion of additional genes encoding for catalase (cat(++)) or eNOS nor eNOS knock-out changed EPCs in resting mice. In striking contrast, inhibition of catalase by aminotriazole strongly reduced circulating EPCs in sedentary cat(++) and their transgen-negative littermates (cat(n)), while forced or voluntary exercise training of cat(++) mice significantly increased the number of circulating EPCs. The latter effect was completely inhibitable by aminotriazole. These data suggest that endogenous vascular H(2)O(2) likely contributes to the impairment of important stem cell-induced vascular repair mechanisms in cardiovascular disease.

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

    Science.gov (United States)

    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.

  13. Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture

    Czech Academy of Sciences Publication Activity Database

    Svobodová, Z.; Kučerová, J.; Autebert, J.; Horák, Daniel; Bruckova, L.; Viovy, J.-L.; Bílková, Z.

    2014-01-01

    Roč. 35, 2-3 (2014), s. 323-329 ISSN 0173-0835 R&D Projects: GA MŠk 7E09109 EU Projects: European Commission(XE) 228980 - CAMINEMS Institutional support: RVO:61389013 Keywords : biofunctionalization * circulating tumor cells * EpCAM Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.028, year: 2014

  14. Low Number of Detectable Circulating Tumor Cells in Non-metastatic Colon Cancer

    DEFF Research Database (Denmark)

    Thorsteinsson, Morten; Söletormos, György; Jess, Per

    2011-01-01

    The aim of the present study was to detect circulating tumor cells (CTCs) in the peripheral blood of patients with non-metastatic colon cancer and to evaluate whether there is a diurnal variation in the CTC counts. Furthermore, the study aimed to examine the correlation between CTCs and TNM stage...

  15. Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer

    NARCIS (Netherlands)

    Alonso-Alconada, Lorena; Muinelo-Romay, Laura; Madissoo, Kadri; Diaz-Lopez, Antonio; Krakstad, Camilla; Trovik, Jone; Wik, Elisabeth; Hapangama, Dharani; Coenegrachts, Lieve; Cano, Amparo; Gil-Moreno, Antonio; Chiva, Luis; Cueva, Juan; Vieito, Maria; Ortega, Eugenia; Mariscal, Javier; Colas, Eva; Castellvi, Josep; Cusido, Maite; Dolcet, Xavier; Nijman, Hans W.; Bosse, Tjalling; Green, John A.; Romano, Andrea; Reventos, Jaume; Lopez-Lopez, Rafael; Salvesen, Helga B.; Amant, Frederic; Matias-Guiu, Xavier; Moreno-Bueno, Gema; Abal, Miguel

    2014-01-01

    Background: About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in

  16. Gene expression profiling in circulating cells (ctcs) of breast carcinoma patients

    Czech Academy of Sciences Publication Activity Database

    Kološtová, K.; Pinterová, D.; Tesařová, P.; Mikulová, V.; Kubecová, M.; Brychta, M.; Rusňáková, Vendula; Kasimir-Bauer, S.; Kubista, Mikael

    2010-01-01

    Roč. 21, suppl. 4 (2010), s. 49-59 ISSN 0923-7534 Institutional research plan: CEZ:AV0Z50520701 Keywords : Circulating tumor cells * Breast cancer * Gene expression profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.452, year: 2010

  17. The effect of local breast radiotherapy on circulating CD34+ cells

    International Nuclear Information System (INIS)

    Alajez, Nehad M.; Wang Wei; Biswas, Debashis; Teh, Amy; Sutherland, Robert; Pintilie, Melania; Minden, Mark; Messner, Hans; Fyles, Anthony; Gospodarowicz, Mary; Keating, Armand; Liu, Fei-Fei

    2011-01-01

    The number of circulating CD34 + (hematopoietic stem) cells (HSCs) was observed to decline by 15% in breast cancer patients after starting adjuvant radiation therapy, regardless of age or preceding chemotherapy. These data demonstrate that local radiation therapy can profoundly affect HSC homeostasis, which might have a myriad of important implications.

  18. Circulating miRNAs as biomarkers for oral squamous cell carcinoma recurrence in operated patients

    DEFF Research Database (Denmark)

    Yan, Yan; Wang, Xuan; Venø, Morten Trillingsgaard

    2017-01-01

    MicroRNAs (miRNAs) are small regulatory non-coding RNAs for which altered expression in cancers can serve as potential biomarkers for diseases. We here investigated whether circulating miRNAs can serve as biomarkers for predicting post-operational recurrence of oral squamous cell carcinoma (OSCC...

  19. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  20. Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Hansen, Jakob Werner; Kristensen, Søren Sommer

    2016-01-01

    III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after......BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients...... treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter. CONCLUSIONS: Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating...

  1. The Repeated Administration of Resveratrol Has Measurable Effects on Circulating T-Cell Subsets in Humans

    Directory of Open Access Journals (Sweden)

    J. Luis Espinoza

    2017-01-01

    Full Text Available Preclinical studies have shown that resveratrol exerts immunomodulatory effects with potential clinical value in the amelioration of autoimmune disorders and cancer prevention; however, little is known about the in vivo effects of this naturally occurring polyphenol on human immune cells. We assessed the effects of repeated doses of resveratrol (1000 mg/day for 28 days on circulating immune cells in healthy Japanese individuals. Resveratrol was safe and well tolerated and was associated with significant increases in the numbers of circulating γδ T cells and regulatory T cells and resulted in small, yet significant, decreases in the plasma levels of the proinflammatory cytokines TNF-α and MCP-1 and a significant increase in the plasma antioxidant activity compared with the corresponding antioxidant baseline activity and with that in four control individuals. In in vitro studies, resveratrol significantly improved the growth of γδ T cells and regulatory T cells. These findings demonstrate that resveratrol has some clear biological effects on human circulating immune cells. Further studies are necessary to interpret the long-term immunological changes associated with resveratrol treatment.

  2. KRAS mutational status analysis of peripheral blood isolated circulating tumor cells in metastatic colorectal patients

    OpenAIRE

    GUTI?RREZ, CRISTINA; RODRIGUEZ, JAVIER; PATI?O-GARC?A, ANA; GARC?A-FONCILLAS, JES?S; SALGADO, JOSEFA

    2013-01-01

    The present study describes an optimized method for isolating peripheral blood circulating tumor cells (CTCs) and performing KRAS mutation analysis. The approach combines isolation of peripheral blood mononuclear cells and immunomagnetic labeling with CD45 and CD326 human microbeads with KRAS analysis performed with a Therascreen KRAS kit by quantitative PCR. KRAS mutations were detected in the CTCs of patients with metastatic colorectal cancer (mCRC). CTCs may represent an alternative to inv...

  3. High level of chromosomal instability in circulating tumor cells of ROS1-rearranged non-small-cell lung cancer

    OpenAIRE

    Pailler, E.; Auger, N.; Lindsay, C. R.; Vielh, P.; Islas-Morris-Hernandez, A.; Borget, I.; Ngo-Camus, M.; Planchard, D.; Soria, J.-C.; Besse, B.; Farace, F.

    2015-01-01

    Background Genetic aberrations affecting the c-ros oncogene 1 (ROS1) tyrosine kinase gene have been reported in a small subset of patients with non-small-cell lung cancer (NSCLC). We evaluated whether ROS1-chromosomal rearrangements could be detected in circulating tumor cells (CTCs) and examined tumor heterogeneity of CTCs and tumor biopsies in ROS1-rearranged NSCLC patients. Patients and methods Using isolation by size of epithelial tumor cells (ISET) filtration and filter-adapted-fluoresce...

  4. Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice.

    Science.gov (United States)

    Höchst, Bastian; Schildberg, Frank A; Böttcher, Jan; Metzger, Christina; Huss, Sebastian; Türler, Andreas; Overhaus, Markus; Knoblich, Andreas; Schneider, Berthold; Pantelis, Dimitrios; Kurts, Christian; Kalff, Jörg C; Knolle, Percy; Diehl, Linda

    2012-11-01

    Immunity against cancer is impeded by local mechanisms promoting development of tumor-specific T cell tolerance, such as regulatory T cells, myeloid-derived suppressor cells, or immunosuppressive factors in the tumor microenvironment. The release of soluble antigens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, has been investigated for diagnostic purposes, but not for its immunological consequences. Here, we address the question of whether soluble CEA influences tumor-specific immunity. Mice were injected with soluble CEA protein, and CEA-specific CD8 T cells were analyzed for their phenotype and functionality by means of restimulation ex vivo or antitumor efficacy in vivo. We furthermore characterized the CD8 T cell population in peripheral blood mononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. In mice, circulating CEA was preferentially taken up in a mannose receptor-dependent manner and cross-presented by liver sinusoidal endothelial cells, but not dendritic cells, to CD8 T cells. Such systemically circulating CEA promoted tolerization of CEA-specific CD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1. These CD8 T cells were not deleted but were rendered nonresponsive to antigen-specific stimulation and failed to control growth of CEA-expressing tumor cells. These nonresponsive CD8 T cells were phenotypically similar to central memory T cells being CD44(high) CD62L(high) CD25(neg) . We found T cells with a similar phenotype in PBMCs of healthy donors and at increased frequency also in patients with colorectal carcinoma. Our results provide evidence for the existence of an unrecognized tumor immune escape involving cross-presentation of systemically circulating tumor antigens that may influence immunotherapy of cancer. Copyright © 2012 American Association for the Study of Liver Diseases.

  5. Detection, isolation, and capture of circulating breast cancer cells with photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran; Njoroge, Martin; Goldschmidt, Benjamin S.; Gaffigan, Brian; Rood, Kyle; Viator, John A.

    2013-03-01

    According to the CDC, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis, or the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems, significantly worsens the prognosis of any breast cancer patient. In this study, a technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser with a 5 ns pulse at 532 nm is used to interrogate thousands of cells with one pulse as they flow through the beam path. Cells which are pigmented, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to provide pigment. After which, the device is calibrated to demonstrate a single-cell detection limit. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25-45 breast cancer cells per 1 mL of blood. An in vitro photoacoustic flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy, it can also be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  6. Gene expression profiling of circulating CD133+cells of hepatocellular carcinoma patients associated with HCV infection.

    Science.gov (United States)

    Zekri, Abdel-Rahman N; El-Sisi, Enas R; Abdallah, Zeinab F; Ismail, Alaa; Barakat Barakat, Ahmed

    2017-03-01

    Identifying the genetic expression profile of CD133 + cells from HCC patients compared to CD133 + cells from healthy volunteers that may contribute in hepatocarcinogenesis process. Circulating CD133 + cells were sorted from the peripheral blood of HCC patients as well as from healthy volunteers using magnetic activated cell sorting. The differential expression profile of stem cell related genes was performed using the Stem Cell PCR profiling assay. Data analysis of stem cells related genes in CD133 + cells of the HCC group compared to the control group showed that; CCND2, COL1A1, CTNNA1, DLL3, JAG1, KRT15, MYC, NOTCH2, T and TERT were up-regulated (fold change=80, 68.6, 6.67, 7.22, 3.8, 15.2, 14.5, 105.6, 26.6 and 99 respectively while only CD3D was down-regulated (fold change=0.055) in HCC patients. However, after application of Beferroni correction to adjust P-value; KRT15 was the only gene that was significantly over expressed in CD133 + cells of HCC compared to control group (P-value=0.012). KRT15 can be used to differentiate between circulating CD133 + cells from HCC group and control group. However, further study may be needed to confirm on the protein level. Copyright © 2016 National Cancer Institute, Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

  7. Circulating Cell-Free DNA Differentiates Severity of Inflammation.

    Science.gov (United States)

    Frank, Mayu O

    2016-10-01

    As the U.S. population ages, the incidence of chronic disease will rise. Chronic diseases have been linked to chronic inflammation. The purpose of this review is to summarize the literature on cell-free DNA (cfDNA) in relation to inflammation. PubMed, EMBASE, and Web of Science were searched. Inclusion criteria were noninterventional studies on acute and chronic inflammation, autoimmunity, and infection published in English after 2000, conducted in humans using the fluorescence method of quantifying DNA. Of the 442 articles retrieved, 83 were identified for full-text review and 13 remained after application of inclusion criteria. Of the reviewed studies, three involved acute inflammation, six involved chronic inflammation, and four involved infection. Healthy controls with interpretable results were included in six studies, three of which used the Quant-iT high-sensitivity DNA kit and found cfDNA quantities near 800 ng/ml, while the other three used other fluorescence methods and found quantities below 100 ng/ml. All 13 studies compared groups, and all but 1 found statistically significant differences between them. Among studies using the Quant-iT reagent, levels were higher in infection than in chronic inflammation. Among studies that used other reagents, levels increased from chronic to acute inflammation to severe infection. CfDNA levels were associated with mortality and with clinical outcomes in acute inflammation and infection. Most studies assessed cfDNA's correlation with other inflammation biomarkers and found inconclusive results. There appears to be an association between inflammation and cfDNA. Further research is necessary before cfDNA can be used clinically as a measure of inflammation. © The Author(s) 2016.

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

    Science.gov (United States)

    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.

  9. Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Thiago, Leandro S; Perez-Andres, Martin; Balanzategui, Ana; Sarasquete, Maria E; Paiva, Bruno; Jara-Acevedo, Maria; Barcena, Paloma; Sanchez, Maria Luz; Almeida, Julia; González, Marcos; San Miguel, Jesus F; Garcia-Sanz, Ramón; Orfao, Alberto

    2014-01-01

    The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/μL (range: 0.0003-0.08 cells/μL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

  10. Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, Olga V. [Creatv MicroTech, Inc., 2242 West Harrison St., Chicago 60612, IL (United States); Adams, Daniel L., E-mail: dan@creatvmicrotech.com [Creatv MicroTech, Inc., 1 Deer Park Drive, Monmouth Junction, NJ 08852 (United States); Divan, Ralu; Rosenmann, Daniel [Center for Nanoscale Materials, Argonne National Laboratory, 9700 South Cass Ave., Argonne 60439, IL (United States); Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei [Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac 20854, MD (United States)

    2016-09-01

    There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture.

  11. Detection of mycoplasma infection in circulating tumor cells in patients with hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hong Seo; Lee, Hyun Min; Kim, Won-Tae; Kim, Min Kyu [Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul (Korea, Republic of); Chang, Hee Jin [Center for Colorectal Cancer, Research Institute and Hospital of National Cancer Center, Goyang-si (Korea, Republic of); Lee, Hye Ran [Department of Internal Medicine, Inje University Ilsan Paik Hospital, Goyang-si (Korea, Republic of); Joh, Jae-Won [Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kim, Dae Shick, E-mail: oncorkim@skku.edu [Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Ryu, Chun Jeih, E-mail: cjryu@sejong.ac.kr [Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul (Korea, Republic of)

    2014-04-04

    Highlights: • This study generates a monoclonal antibody CA27 against the mycoplasmal p37 protein. • CA27 isolates circulating tumor cells (CTCs) from the blood of liver cancer patients. • Results show the first evidence for mycoplasma infected-CTCs in cancer patients. - Abstract: Many studies have shown that persistent infections of bacteria promote carcinogenesis and metastasis. Infectious agents and their products can modulate cancer progression through the induction of host inflammatory and immune responses. The presence of circulating tumor cells (CTCs) is considered as an important indicator in the metastatic cascade. We unintentionally produced a monoclonal antibody (MAb) CA27 against the mycoplasmal p37 protein in mycoplasma-infected cancer cells during the searching process of novel surface markers of CTCs. Mycoplasma-infected cells were enriched by CA27-conjugated magnetic beads in the peripheral blood mononuclear cells in patients with hepatocellular carcinoma (HCC) and analyzed by confocal microscopy with anti-CD45 and CA27 antibodies. CD45-negative and CA27-positive cells were readily detected in three out of seven patients (range 12–30/8.5 ml blood), indicating that they are mycoplasma-infected circulating epithelial cells. CA27-positive cells had larger size than CD45-positive hematological lineage cells, high nuclear to cytoplasmic ratios and irregular nuclear morphology, which identified them as CTCs. The results show for the first time the existence of mycoplasma-infected CTCs in patients with HCC and suggest a possible correlation between mycoplasma infection and the development of cancer metastasis.

  12. Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

    Science.gov (United States)

    Geislinger, Thomas M.; Stamp, Melanie E. M.; Wixforth, Achim; Franke, Thomas

    2015-11-01

    We demonstrate continuous, passive, and label-free sorting of different in vitro cancer cell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumor cells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples.

  13. High frequency of circulating ¿d T cells with dominance of the vd1 subset in a healthy population

    DEFF Research Database (Denmark)

    Hviid, L; Akanmori, B D; Loizon, S

    2000-01-01

    TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequenc...

  14. The clinical significance of circulating tumor cells in non-metastatic colorectal cancer - A review

    DEFF Research Database (Denmark)

    Thorsteinsson, M; Jess, Per

    2011-01-01

    BACKGROUND: Finding a clinical tool to improve the risk stratification and identifying those colorectal cancer patients with an increased risk of recurrence is of great importance. The presence of circulating tumor cells (CTC) in peripheral blood can be a strong marker of poor prognosis in patients...... with metastatic disease, but the prognostic role of CTC in non-metastatic colorectal cancer is less clear. The aim of this review is to examine the possible clinical significance of circulating tumor cells in non-metastatic colorectal cancer (TNM-stage I-III) with the primary focus on detection methods...... and prognosis. METHODS: The PubMed and Cochrane database and reference lists of relevant articles were searched for scientific literature published in English from January 2000 to June 2010. We included studies with non-metastatic colorectal cancer (TNM-stage I-III) and CTC detected pre- and/or post...

  15. Detection of circulating breast tumor cells by differential expression of marker genes

    NARCIS (Netherlands)

    Bosma, Astrid J.; Weigelt, Britta; Lambrechts, A. Caro; Verhagen, Onno J. H. M.; Pruntel, Roelof; Hart, Augustinus A. M.; Rodenhuis, Sjoerd; van 't Veer, Laura J.

    2002-01-01

    Purpose: We undertook a systematic approach to identify breast cancer (BC) marker genes with molecular assays and evaluated these marker genes for the detection of minimal residual disease in peripheral blood mononuclear cells (PBMCs). Experimental Design: We used serial analysis of gene expression

  16. Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry.

    Science.gov (United States)

    Au, Sam H; Edd, Jon; Stoddard, Amy E; Wong, Keith H K; Fachin, Fabio; Maheswaran, Shyamala; Haber, Daniel A; Stott, Shannon L; Kapur, Ravi; Toner, Mehmet

    2017-05-26

    Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2-100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

  17. Biodegradable nano-films for capture and non-invasive release of circulating tumor cells

    Science.gov (United States)

    Park, Myoung-Hwan; Castleberry, Steven; Deng, Jason Z.; Hsu, Bryan; Mayner, Sarah; Jensen, Anne E.; Sequist, Lecia V.; Maheswaran, Shyamala; Haber, Daniel A.; Toner, Mehmet; Stott, Shannon L.; Hammond, Paula T.

    2016-01-01

    Selective isolation and purification of circulating tumor cells (CTCs) from whole blood is an important capability for both clinical medicine and biological research. Current techniques to perform this task place the isolated cells under excessive stresses that reduce cell viability, and potentially induce phenotype change, therefore losing valuable information about the isolated cells. We present a biodegradable nano-film coating on the surface of a microfluidic chip, which can be used to effectively capture as well as non-invasively release cancer cell lines such as PC-3, LNCaP, DU 145, H1650 and H1975. We have applied layer-by-layer (LbL) assembly to create a library of ultrathin coatings using a broad range of materials through complementary interactions. By developing an LbL nano-film coating with an affinity-based cell-capture surface that is capable of selectively isolating cancer cells from whole blood, and that can be rapidly degraded on command, we are able to gently isolate cancer cells and recover them without compromising cell viability or proliferative potential. Our approach has the capability to overcome practical hurdles and provide viable cancer cells for downstream analyses, such as live cell imaging, single cell genomics, and in vitro cell culture of recovered cells. Furthermore, CTCs from cancer patients were also captured, identified, and successfully released using the LbL-modified microchips. PMID:26142780

  18. Probing Androgen Receptor Signaling in Circulating Tumor Cells in Prostate Cancer

    Science.gov (United States)

    2013-07-01

    membrane antigen . Proc Natl Acad Sci U S A 2011 ; 108 : 9578 – 82 . 9. Danila DC , Heller G , Gignac GA , Gonzalez -Espinoza R , Anand...13 : 7053 – 8 . 10. Helo P , Cronin AM , Danila DC , Wenske S , Gonzalez -Espinoza R , Anand A , et al. Circulating prostate tumor...mesenchymal transition. J. Clin. Invest. 119, 1420–1428 (2009). 13. O. Lara , X. Tong, M. Zborowski, J. J. Chalmers, Enrichment of rare cancer cells

  19. Deterministic Capture of Individual Circulating Tumor Cells Using a Flow-Restricted Microfluidic Trap Array

    Directory of Open Access Journals (Sweden)

    Yousang Yoon

    2018-03-01

    Full Text Available Circulating tumor cells (CTCs are regarded as a strong biomarker which includes clinically valuable information. However, CTCs are very rare and require precise separation and detection for effective clinical applications. Furthermore, downstream analysis has become necessary to identify the distinct sub-population of CTCs that causes metastasis. Here, we report a flow-restricted microfluidic trap array capable of deterministic single-cell capture of CTCs. The extent of flow restriction, correlating with the device geometry, was then optimized using a highly invasive breast cancer cell line (LM2 MDA-MB-231 to achieve 97% capture efficiency with a single-cell capture rate of 99%. Single-cell capture of CTCs from mice with full-blown metastasis was also demonstrated. The single-CTC capturing ability of the flow-restricted trap array not only showed cell enumerating ability but also high prospects for application in future automated downstream analysis.

  20. Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

    Directory of Open Access Journals (Sweden)

    C.R.T. Godoy

    2015-06-01

    Full Text Available We measured circulating endothelial precursor cells (EPCs, activated circulating endothelial cells (aCECs, and mature circulating endothelial cells (mCECs using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML patients and 65 healthy controls; and vascular endothelial growth factor (VEGF by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146% was significantly higher than in healthy subjects (0.0059%, P0.05. In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145. In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.

  1. Long circulating micelles of an amphiphilic random copolymer bearing cell outer membrane phosphorylcholine zwitterions.

    Science.gov (United States)

    Zhao, Jing; Chai, Yu-Dong; Zhang, Jing; Huang, Peng-Fei; Nakashima, Kenichi; Gong, Yong-Kuan

    2015-04-01

    Polymeric micelles with cell outer membrane mimetic structure were prepared in water from amphiphilic random copolymers bearing both the hydrophilic phosphorylcholine zwitterions and hydrophobic octadecyl side chains of cell outer membrane. The polymeric micelles showed sizes ranging from 80 nm to 120 nm in hydrodynamic diameter and zeta-potentials from -6.4 mV to -2.4 mV by dynamic light scattering measurements. The micelles loaded with 6-coumarin as a fluorescence probe were stable to investigate their blood circulation and biodistribution. The in vitro phagocytosis results using murine peritoneal macrophages showed 10-fold reduction compared with a reference micelle. The in vivo blood circulation half-life of the polymeric micelles following intravenous administration in New Zealand Rabbits was increased from 0.55 h to 90.5h. More interestingly, tissue distribution results showed that the concentration of the micelles in the kidney is 4-fold higher than that in the liver and other organs 48 h after administration. The results of this work show great promise for designing more effective stealth drug carriers that can minimize reticuloendothelial system clearance and circulate for long time to reach target by using simple cell membrane mimetic random copolymer micelles. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. Chemically Modified Plastic Tube for High Volume Removal and Collection of Circulating Tumor Cells.

    Directory of Open Access Journals (Sweden)

    Angelo Gaitas

    Full Text Available In this preliminary effort, we use a commercially available and chemically modified tube to selectively capture circulating tumor cells (CTCs from the blood stream by immobilizing human anti-EpCAM antibodies on the tube's interior surface. We describe the requisite and critical steps required to modify a tube into a cancer cell-capturing device. Using these simple modifications, we were able to capture or entrap about 85% of cancer cells from suspension and 44% of cancer cells from spiked whole blood. We also found that the percentage of cells captured was dependent on the tube's length and also the number of cancer cells present. It is our strong belief that with the utilization of appropriate tube lengths and procedures, we can ensure capture and removal of nearly the entire CTC population in whole blood. Importantly after a patient's entire blood volume has circulated through the tube, the tube can then be trypsinized to release the captured live CTCs for further analysis and testing.

  3. Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells

    Science.gov (United States)

    Herati, Ramin Sedaghat; Muselman, Alexander; Vella, Laura; Bengsch, Bertram; Parkhouse, Kaela; Del Alcazar, Daniel; Kotzin, Jonathan; Doyle, Susan A.; Tebas, Pablo; Hensley, Scott E.; Su, Laura F.; Schmader, Kenneth E.; Wherry, E. John

    2017-01-01

    T follicular helper (Tfh) CD4 cells are crucial providers of B cell help during adaptive immune responses. A circulating population of CD4 T cells, termed cTfh, have similarity to lymphoid Tfh, can provide B cell help, and responded to influenza vaccination. However, it is unclear whether human vaccination-induced cTfh respond in an antigen-specific manner and whether they form long-lasting memory. Here, we identified a cTfh population that expressed multiple T cell activation markers and could be readily identified by coexpression of ICOS and CD38. This subset expressed more Bcl-6, c-Maf, and IL-21 than other blood CD4 subsets. Influenza vaccination induced a strong response in the ICOS+CD38+ cTfh at day 7, and this population included hemagglutinin-specific cells by tetramer staining and antigen-stimulated Activation Induced Marker (AIM) expression. Moreover, TCRB sequencing identified a clonal response in ICOS+CD38+ cTfh that correlated strongly with the increased circulating ICOS+CD38+ cTfh frequency and the circulating plasmablast response. In subjects who received successive annual vaccinations, a recurrent oligoclonal response was identified in the ICOS+CD38+ cTfh subset at 7 days after every vaccination. These oligoclonal responses in ICOS+CD38+ cTfh after vaccination persisted in the ICOS−CD38− cTfh repertoire in subsequent years, suggesting clonal maintenance in a memory reservoir in the more-stable ICOS−CD38− cTfh subset. These data highlight the antigen-specificity, lineage relationships and memory properties of human cTfh responses to vaccination, providing new avenues for tracking and monitoring cTfh responses during infection and vaccination in humans. PMID:28620653

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

    2016-03-29

    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.

  6. Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

    Directory of Open Access Journals (Sweden)

    Teresa Anglada

    2016-01-01

    Full Text Available In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated defective cell line, as Ataxia-Telangiectasia (AT cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative carried ≥10 γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

  7. Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells

    KAUST Repository

    Malara, Natalia

    2014-07-01

    Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor\\'s stadiation, therapy, and early relapsing lesions. Within surface\\'s bio-functionalization and cell\\'s isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient\\'s blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Improved Detection of Circulating Epithelial Cells in Patients with Intraductal Papillary Mucinous Neoplasms.

    Science.gov (United States)

    Franses, Joseph W; Basar, Omer; Kadayifci, Abdurrahman; Yuksel, Osman; Choz, Melissa; Kulkarni, Anupriya S; Tai, Eric; Vo, Kevin D; Arora, Kshitij S; Desai, Niyati; Licausi, Joseph A; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A; Ryan, David P; Brugge, William R; Ting, David T

    2018-01-01

    Recent work has demonstrated early shedding of circulating epithelial cells (CECs) from premalignant intraductal papillary mucinous neoplasms (IPMNs). However, the potential use of CECs as a "liquid biopsy" for patients with IPMNs has been limited by antigen dependence of CEC isolation devices and the lack of robust detection biomarkers across CEC phenotypes. We utilized a negative depletion microfluidic platform to purify CECs from contaminating leukocytes and coupled this platform with immunofluorescence, RNA in situ hybridization, and RNA sequencing (RNA-seq) detection and enumeration. Using established protein (EpCAM, cytokeratins) and novel noncoding RNA (HSATII, cytokeratins) biomarkers, we detected CECs in 88% of patients bearing IPMN lesions. RNA-seq analysis for MUC genes confirm the likely origin of these CECs from pancreatic lesions. Our findings increase the sensitivity of detection of these cells and therefore could have clinical implications for cancer risk stratification. This work describes a high-sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at high risk of malignant transformation. Further research efforts are underway to define the transcriptional programs that might allow discrimination between circulating cells released from tumors that will become malignant and cells released from tumors that will not. After further refinement, this combination of technologies could be deployed for monitoring and early detection of patients at high risk for developing new or recurrent pancreatic malignancies. © AlphaMed Press 2017.

  9. Imaging circulating tumor cells in freely moving awake small animals using a miniaturized intravital microscope.

    Directory of Open Access Journals (Sweden)

    Laura Sarah Sasportas

    Full Text Available Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals.

  10. Detection and cultivation of circulating tumor cells in malignant pleural mesothelioma.

    Science.gov (United States)

    Bobek, Vladimir; Kacprzak, Grzegorz; Rzechonek, Adam; Kolostova, Katarina

    2014-05-01

    Malignant pleural mesothelioma (MPM) is an aggressive disease with very poor prognosis which tends to affect older patients. Progress in the management of this group of patients has been limited by the rarity of the disease and hence, difficulty in conducting randomized trials. The vast majority of cancer deaths occur due to metastasis of the primary tumor to distant sites via circulating tumor cells (CTCs) in the circulation. CTCs are extremely rare and limits in technology used to capture these cells hamper our complete understanding over the metastatic process. In the present study we present a new method for detection and cultivation of CTCs isolated from peripheral blood of MPM patients. Patients with diagnosed MPM were enrolled into this study. A size-based separation method for viable CTC enrichment from unclothed peripheral blood has been introduced; MetaCell. The size-based enrichment process was based on filtration of peripheral blood (PB) through porous polycarbonate membrane. The separated CTCs are cultured on the membrane in vitro under standard cancer cell culture conditions and observed by an inverted microscope. The reported methodology allows for quick and easy enrichment of CTCs and their cultivation. The cultivated cells can be used for next specification of gene expression and histological/biological specificity of concrete mesothelioma.

  11. In vitro detection of circulating tumor cells compared by the CytoTrack and CellSearch methods

    DEFF Research Database (Denmark)

    Hillig, T.; Horn, P.; Nygaard, Ann-Britt

    2015-01-01

    Comparison of two methods to detect circulating tumor cells (CTC) CytoTrack and CellSearch through recovery of MCF-7 breast cancer cells, spiked into blood collected from healthy donors. Spiking of a fixed number of EpCAM and pan-cytokeratin positive MCF-7 cells into 7.5 mL donor blood...... were determined. The average numbers of MCF-7 cells/cells in clusters/clusters recovered from blood by the CytoTrack and CellSearch methods were 103 +/- 5.9/27 +/- 7.9/11 +/- 3.5 (95 % CI) and 107 +/- 4.4/20 +/- 7.1/10 +/- 3.5, respectively, with no difference between the two methods (p = 0.37/p = 0...

  12. Diurnal Variations of Human Circulating Cell-Free Micro-RNA

    DEFF Research Database (Denmark)

    Heegaard, Niels H H; Carlsen, Anting Liu; Lilje, Berit

    2016-01-01

    . An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA) that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue...... expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation...... expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples...

  13. Circulating concentrations of stem-cell-mobilizing cytokines are associated with levels of osteoprogenitor cells and aortic calcification severity

    International Nuclear Information System (INIS)

    Pal, S.N.; Clancy, P.; Golledge, J.

    2011-01-01

    The background of this study was to investigate the association between aortic calcification, concentrations of stem-cell-mobilizing cytokines and osteocalcin-positive mononuclear cells in a mouse model and patients with peripheral artery disease. We estimated the concentration of the stem-cell-mobilizing cytokines stromal cell-derived factor α (SDF-1α), granulocyte colony stimulating factor and stem cell factor in a mouse model of aortic calcification developed in osteoprotegerin-deficient (OPG -/- ) mice, as well as in patient plasma samples. Calcification was estimated by a colorimetric assay of extracts of harvested mice aortas and by computed tomographic angiogram images in patients. The cytokine concentrations were assessed for association with the severity of calcification and the percentage of osteocalcin-positive mononuclear cells (OCN + MNC) using non-parametric analysis. The serum concentration of stromal SDF-1α and granulocyte-colony stimulating factor (G-CSF) were significantly greater in OPG -/- compared to control mice. The percentage of circulating OCN + MNC was correlated to the concentration of SDF-1α in OPG -/- mice. These cytokines also correlated with the severity of calcification in OPG -/- mice. Patients with more severe calcification had a higher plasma concentration of the cytokines than those with less marked aortic calcification. The concentrations also correlated with circulating OCN + MNC and aortic calcification volumes. The association between stem cell cytokines, OCN + MNC and calcification suggests a possible role of bone-derived osteoprogenitor cells in the pathogenesis of vascular calcification. (author)

  14. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    Science.gov (United States)

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-04-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

  15. Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

    Science.gov (United States)

    Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B.; Goldman, Jonathan; Rao, Jianyu; Sledge, George W.; Pegram, Mark D.; Sheth, Shruti; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

    2016-01-01

    Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

  16. Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives.

    Science.gov (United States)

    Cima, Igor; Wen Yee, Chay; Iliescu, Florina S; Phyo, Wai Min; Lim, Kiat Hon; Iliescu, Ciprian; Tan, Min Han

    2013-01-01

    This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to "marker-based" techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics.

  17. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    International Nuclear Information System (INIS)

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer

  18. Profiling protein expression in circulating tumour cells using microfluidic western blotting.

    Science.gov (United States)

    Sinkala, Elly; Sollier-Christen, Elodie; Renier, Corinne; Rosàs-Canyelles, Elisabet; Che, James; Heirich, Kyra; Duncombe, Todd A; Vlassakis, Julea; Yamauchi, Kevin A; Huang, Haiyan; Jeffrey, Stefanie S; Herr, Amy E

    2017-03-23

    Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

  19. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    Energy Technology Data Exchange (ETDEWEB)

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  20. Benchtop Technologies for Circulating Tumor Cells Separation Based on Biophysical Properties

    Directory of Open Access Journals (Sweden)

    Wan Shi Low

    2015-01-01

    Full Text Available Circulating tumor cells (CTCs are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies.

  1. Circulating Tumor Cells Detection and Counting in Uveal Melanomas by a Filtration-Based Method

    International Nuclear Information System (INIS)

    Mazzini, Cinzia; Pinzani, Pamela; Salvianti, Francesca; Scatena, Cristian; Paglierani, Milena; Ucci, Francesca; Pazzagli, Mario; Massi, Daniela

    2014-01-01

    Uveal melanoma is one of the most deadly diseases in ophthalmology for which markers able to predict the appearance of metastasis are needed. The study investigates the role of circulating tumor cells (CTC) as a prognostic factor in this disease. We report the detection of circulating tumor cells by Isolation by Size of Epithelial Tumor cells (ISET) in a cohort of 31 uveal melanoma patients: we identified single CTCs or clusters of cells in 17 patients, while the control population, subjects with choroidal nevi, showed no CTC in peripheral blood. The presence of CTCs did not correlate with any clinical and pathological parameter, such as tumor larger basal diameter (LBD), tumor height and TNM. By stratifying patients in groups on the basis of the number of CTC (lower or higher than 10 CTC per 10 mL blood) and the presence of CTC clusters we found a significant difference in LBD (p = 0.019), Tumor height (p = 0.048), disease-free and overall survival (p < 0.05). In conclusion, we confirm the role of CTC as a negative prognostic marker in uveal melanoma patients after a long follow-up period. Further characterization of CTC will help understanding uveal melanoma metastasization and improve patient management

  2. Fluorescence detection, enumeration and characterization of single circulating cells in vivo: technology, applications and future prospects

    Science.gov (United States)

    Hartmann, Carolin; Patil, Roshani; Lin, Charles P.; Niedre, Mark

    2018-01-01

    There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immunology, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations. In contrast, ‘in vivo flow cytometry’ (IVFC) refers to set of technologies that allow study of cells directly in the bloodstream of the organism in vivo. In recent years the IVFC field has grown significantly and new techniques have been developed, including fluorescence microscopy, multi-photon, photo-acoustic, and diffuse fluorescence IVFC. In this paper we review recent technical advances in IVFC, with emphasis on instrumentation, contrast mechanisms, and detection sensitivity. We also describe key applications in biomedical research, including cancer research and immunology. Last, we discuss future directions for IVFC, as well as prospects for broader adoption by the biomedical research community and translation to humans clinically.

  3. A rare case of extremely high counts of circulating tumor cells detected in a patient with an oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Wu, Xianglei; Mastronicola, Romina; Tu, Qian; Faure, Gilbert Charles; De Carvalho Bittencourt, Marcelo; Dolivet, Gilles

    2016-01-01

    Despite aggressive regimens, the clinical outcome of head and neck squamous cell carcinoma remains poor. The detection of circulating tumor cells could potentially improve the management of patients with disseminated cancer, including diagnosis, treatment strategies, and surveillance. Currently, CellSearch ® is the most widely used and the only Food and Drug Administration-cleared system for circulating tumor cells detection in patients with metastatic breast, colorectal, or prostate cancer. In most cases of head and neck squamous cell carcinoma, only low counts of circulating tumor cells have been reported. A 56-year-old white male with no particular medical history, was diagnosed with a squamous cell carcinoma of oral cavity. According to the imaging results (computed tomography and 18 F-fluorodeoxyglucose positron emission tomography / computed tomography) and panendoscopy, the TNM staging was classified as T4N2M0. A non-interruptive pelvimandibulectomy was conducted according to the multidisciplinary meeting advices and the postoperative observations were normal. The patient complained of a painful cervical edema and a trismus 6 weeks after the surgery. A relapse was found by computed tomography and the patient died two weeks later. The search for circulating tumor cells in peripheral venous blood by using the CellSearch ® system revealed a very high count compared with published reports at three time points (pre-operative: 400; intra-operative: 150 and post-operative day 7: 1400 circulating tumor cells). Of note, all detected circulating tumor cells were epidermal growth factor receptor negative. We report here for the first time a rare case of oral squamous cell carcinoma with extremely high circulating tumor cells counts using the CellSearch ® system. The absolute number of circulating tumor cells might predict a particular phase of cancer development as well as a poor survival, potentially contributing to a personalized healthcare

  4. Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

    Science.gov (United States)

    Matsui, Ken; Adelsberger, Joseph W.; Kemp, Troy J.; Baseler, Michael W.; Ledgerwood, Julie E.; Pinto, Ligia A.

    2015-01-01

    Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like) cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as

  5. Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes

    Directory of Open Access Journals (Sweden)

    Michael J. Mitchell

    2012-01-01

    Full Text Available Hematogenous metastasis, the process of cancer cell migration from a primary to distal location via the bloodstream, typically leads to a poor patient prognosis. Selectin proteins hold promise in delivering drug-containing nanocarriers to circulating tumor cells (CTCs in the bloodstream, due to their rapid, force-dependent binding kinetics. However, it is challenging to deliver such nanocarriers while avoiding toxic effects on healthy blood cells, as many possess ligands that adhesively interact with selectins. Herein, we describe a nanostructured surface to capture flowing cancer cells, while preventing human neutrophil adhesion. Microtube surfaces with immobilized halloysite nanotubes (HNTs and E-selectin functionalized liposomal doxorubicin (ES-PEG L-DXR significantly increased the number of breast adenocarcinoma MCF7 cells captured from flow, yet also significantly reduced the number of captured neutrophils. Neutrophils firmly adhered and projected pseudopods on surfaces coated only with liposomes, while neutrophils adherent to HNT-liposome surfaces maintained a round morphology. Perfusion of both MCF7 cells and neutrophils resulted in primarily cancer cell adhesion to the HNT-liposome surface, and induced significant cancer cell death. This work demonstrates that nanostructured surfaces consisting of HNTs and ES-PEG L-DXR can increase CTC recruitment for chemotherapeutic delivery, while also preventing healthy cell adhesion and uptake of therapeutic intended for CTCs.

  6. Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection.

    Science.gov (United States)

    Shi, Jian; Luo, Fengbao; Shi, Qianqian; Xu, Xianlin; He, Xiaozhou; Xia, Ying

    2015-11-03

    Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study. The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules including chemokine receptor type 5 (CXCR5), inducible T cell co-stimulator (ICOS), programmed death-1 (PD-1), ICOSL, PDL-1 and interleukin-21 (IL-21), of peripheral blood were comparatively measured in 42 primary renal allograft recipients within 1-3 years after transplantation. Among them, 24 patients had definite chronic rejection, while other 18 patients had normal renal function. Tfh-cell ratio was significantly increased with PD-1 down-regulation in the patients with chronic renal allograft rejection, while B cells and the alloimmune-regulating molecules studied did not show any appreciable change in parallel. The patients with chronic renal allograft rejection have a characteristic increase in circulating Tfh cells with a decrease in PD-1 expression. These pathological changes may be a therapeutic target for the treatment of chronic renal allograft rejection and can be useful as a clinical index for monitoring conditions of renal transplant.

  7. Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu; Rosenmann, Daniel; Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei

    2016-09-01

    There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. Copyright 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. An increase in circulating B cell-activating factor in childhood-onset ocular myasthenia gravis.

    Science.gov (United States)

    Motobayashi, Mitsuo; Inaba, Yuji; Nishimura, Takafumi; Kobayashi, Norimoto; Nakazawa, Yozo; Koike, Kenichi

    2015-04-01

    Myasthenia gravis is a B cell-mediated autoimmune disorder. The pathophysiology of childhood-onset ocular myasthenia gravis remains unclear. We investigated serum B cell-activating factor levels and other immunological parameters in child patients with ocular myasthenia gravis. Blood samples were obtained from 9 children with ocular myasthenia gravis and 20 age-matched controls. We assayed serum concentrations of B cell-activating factor, anti-acetylcholine receptor antibody titers, 7 types of cytokines (interleukins-2, -4, -6, -10, and -17A; interferon-γ; tumor necrosis factor-α) as well as the percentages of peripheral blood CD4+, CD8+, and CD19+ cells. Serum B cell-activating factor levels were significantly higher before immunosuppressive therapy in patients with childhood-onset ocular myasthenia gravis than in controls and decreased after immunosuppressive therapy. A significant positive correlation was observed between serum B cell-activating factor levels and anti-acetylcholine receptor antibody titers in patients with myasthenia gravis. Serum B cell-activating factor concentrations did not correlate with the percentages of CD4+, CD8+, and CD19+ cells or the CD4+/CD8+ ratio. No significant differences were observed in the levels of the 7 different types of cytokines examined, including interleukin-17A, between preimmunosuppressive therapy myasthenia gravis patients and controls. Circulating B cell-activating factor may play a key role in the pathophysiology of childhood-onset ocular myasthenia gravis. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Changes in Circulating B Cell Subsets Associated with Aging and Acute SIV Infection in Rhesus Macaques.

    Science.gov (United States)

    Chang, W L William; Gonzalez, Denise F; Kieu, Hung T; Castillo, Luis D; Messaoudi, Ilhem; Shen, Xiaoying; Tomaras, Georgia D; Shacklett, Barbara L; Barry, Peter A; Sparger, Ellen E

    2017-01-01

    Aging and certain viral infections can negatively impact humoral responses in humans. To further develop the nonhuman primate (NHP) model for investigating B cell dynamics in human aging and infectious disease, a flow cytometric panel was developed to characterize circulating rhesus B cell subsets. Significant differences between human and macaque B cells included the proportions of cells within IgD+ and switched memory populations and a prominent CD21-CD27+ unswitched memory population detected only in macaques. We then utilized the expanded panel to analyze B cell alterations associated with aging and acute simian immunodeficiency virus (SIV) infection in the NHP model. In the aging study, distinct patterns of B cell subset frequencies were observed for macaques aged one to five years compared to those between ages 5 and 30 years. In the SIV infection study, B cell frequencies and absolute number were dramatically reduced following acute infection, but recovered within four weeks of infection. Thereafter, the frequencies of activated memory B cells progressively increased; these were significantly correlated with the magnitude of SIV-specific IgG responses, and coincided with impaired maturation of anti-SIV antibody avidity, as previously reported for HIV-1 infection. These observations further validate the NHP model for investigation of mechanisms responsible for B cells alterations associated with immunosenescence and infectious disease.

  10. Tumor Cell Malignant Properties Are Enhanced by Circulating Exosomes in Sleep Apnea.

    Science.gov (United States)

    Almendros, Isaac; Khalyfa, Abdelnaby; Trzepizur, Wojciech; Gileles-Hillel, Alex; Huang, Lei; Akbarpour, Mahzad; Andrade, Jorge; Farré, Ramon; Gozal, David

    2016-11-01

    OSA is associated with increased cancer incidence and mortality. Exosomes are vesicles secreted by most cells. They are released into the bloodstream and play a role in tumor progression and metastasis. We evaluated whether the chronic intermittent hypoxia (IH) that characterizes OSA leads to release of tumor-promoting exosomes in the circulation. C57/B6 male mice were randomized to 6 weeks of IH or room air (RA). A subgroup was injected with TC1 lung carcinoma cells in the left flank after 2 weeks of IH. Exosomes from mouse plasma and from 10 adult human patients with OSA before and after treatment for 6 weeks were cocultured with mouse TC1 and human adenocarcinoma cells lines. Malignant tumor properties such as proliferation, migration, invasion, and endothelial monolayer disruption were assessed, as was micro-RNA (miRNA), exosomal content, and transcriptomic effects of exosomes on TC1 cells in vitro to identify target genes. Application of IH-induced exosomes from either IH-exposed tumor-bearing (IH+) or non-tumor-bearing (IH-) mice significantly promoted TC1 malignant properties. Similarly, before adherent treatment, exosomes from patients with OSA significantly enhanced proliferation and migration of human adenocarcinoma cells compared with after adherent treatment. Eleven distinct miRNAs emerged in IH-exposed mice, and their gene targets in TC1 cells were identified. Circulating exosomes released under IH conditions in vivo selectively enhance specific properties of lung tumor cell cultures. Thus, plasma exosomes participate in the increased tumor aggressiveness observed in patients with OSA. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  11. The presence of local and circulating autoreactive B cells in patients with advanced periodontitis.

    Science.gov (United States)

    Berglundh, Tord; Liljenberg, Birgitta; Tarkowski, Andrej; Lindhe, Jan

    2002-04-01

    The aim of the present investigation was to study the local (gingival) and systemic occurrence of autoreactive B cells (CD5+CD19 positive) in subjects with a high or low susceptibility to periodontitis. 2 groups of subjects (Group A and B) susceptible to periodontitis were included. Group A consisted of 22 adult patients (7 females and 15 males, aged 24-66 years) with advanced and generalized chronic periodontitis and group B comprised 7 children (4 girls and 3 boys aged 9-13 years) with localized aggressive periodontitis. 26 periodontally healthy subjects, Group C (aged 23-80 years, mean 49.6+/-16.3), were also recruited. Assessment of clinical and radiographical characteristics of periodontal disease was performed. Gingival biopsies and peripheral blood samples were obtained and prepared for immunohistochemical analysis. Blood samples only were obtained from the periodontally healthy subjects (group C). The proportion of autoreactive B cells (CD5+CD19 positive) of peripheral blood lymphocytes was about 6 times higher in group A and 4 times higher in group B than in the samples from the control subjects (group C). About 40-50% of the B cells in the peripheral blood of the periodontitis susceptible individuals expressed markers for autoreactive features while less than 15% of the circulating B cells in the subjects of group C exhibited such markers. The periodontitis lesion in the adult periodontitis patients contained a substantial number of B cells out of which about 30% demonstrated autoreactive features. It is suggested that both circulating and local B cells in periodontitis susceptible individuals have a higher propensity to autoreactive properties than B cells of patients with a low susceptibility to periodontitis.

  12. Different Profile of Interleukin-10 Production in Circulating T Cells from Atopic Asthmatics Compared with Healthy Subjects

    Directory of Open Access Journals (Sweden)

    K Matsumoto

    2004-01-01

    Full Text Available BACKGROUND: Interleukin (IL-10 is a pleiotropic cytokine released from various cells, including T cells. Although IL-10 is suggested to inhibit allergic responses, its role in asthma remains uncertain. The purpose of the present study was to compare the profile of IL-10 in circulating T cells from stable atopic asthmatics, atopic nonasthmatics and healthy controls.

  13. Type 2 diabetes mellitus is associated with an imbalance in circulating endothelial and smooth muscle progenitor cell numbers

    NARCIS (Netherlands)

    van Ark, J.; Moser, J.; Lexis, C. P. H.; Bekkema, F.; Pop, I.; van der Horst, I. C. C.; Zeebregts, C. J.; van Goor, H.; Wolffenbuttel, B. H. R.; Hillebrands, J. L.

    Individuals with type 2 diabetes mellitus have increased rates of macrovascular disease (MVD). Endothelial progenitor cells (EPCs), circulating angiogenic cells (CACs) and smooth muscle progenitor cells (SMPCs) are suggested to play a role in the pathogenesis of MVD. The relationship between

  14. In vivo Ebola virus infection leads to a strong innate response in circulating immune cells.

    Science.gov (United States)

    Caballero, Ignacio S; Honko, Anna N; Gire, Stephen K; Winnicki, Sarah M; Melé, Marta; Gerhardinger, Chiara; Lin, Aaron E; Rinn, John L; Sabeti, Pardis C; Hensley, Lisa E; Connor, John H

    2016-09-05

    Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells. Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues. Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis.

  15. Ultra-fast vitrification of patient-derived circulating tumor cell lines.

    Science.gov (United States)

    Sandlin, Rebecca D; Wong, Keith H K; Tessier, Shannon N; Swei, Anisa; Bookstaver, Lauren D; Ahearn, Bennett E; Maheswaran, Shyamala; Haber, Daniel A; Stott, Shannon L; Toner, Mehmet

    2018-01-01

    Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

  16. Nanotechnology-Based Strategies for Early Cancer Diagnosis Using Circulating Tumor Cells as a Liquid Biopsy.

    Science.gov (United States)

    Huang, Qinqin; Wang, Yin; Chen, Xingxiang; Wang, Yimeng; Li, Zhiqiang; Du, Shiming; Wang, Lianrong; Chen, Shi

    2018-01-01

    Circulating tumor cells (CTCs) are cancer cells that shed from a primary tumor and circulate in the bloodstream. As a form of "tumor liquid biopsy", CTCs provide important information for the mechanistic investigation of cancer metastasis and the measurement of tumor genotype evolution during treatment and disease progression. However, the extremely low abundance of CTCs in the peripheral blood and the heterogeneity of CTCs make their isolation and characterization major technological challenges. Recently, nanotechnologies have been developed for sensitive CTC detection; such technologies will enable better cell and molecular characterization and open up a wide range of clinical applications, including early disease detection and evaluation of treatment response and disease progression. In this review, we summarize the nanotechnology-based strategies for CTC isolation, including representative nanomaterials (such as magnetic nanoparticles, gold nanoparticles, silicon nanopillars, nanowires, nanopillars, carbon nanotubes, dendrimers, quantum dots, and graphene oxide) and microfluidic chip technologies that incorporate nanoroughened surfaces and discuss their key challenges and perspectives in CTC downstream analyses, such as protein expression and genetic mutations that may reflect tumor aggressiveness and patient outcome.

  17. Manganese effects on haematopoietic cells and circulating coelomocytes of Asterias rubens (Linnaeus)

    Energy Technology Data Exchange (ETDEWEB)

    Oweson, Carolina; Skoeld, Helen [Department of Marine Ecology, Goeteborg University, Kristineberg 566, 45034 Fiskebaeckskil (Sweden); Pinsino, Annalisa; Matranga, Valeria [Istituto di Biomedicina e Immunologia Molecolare ' A. Monroy' , Via Ugo La Malfa 153, 90146 Palermo (Italy); Hernroth, Bodil [The Royal Swedish Academy of Sciences, Kristineberg 566, 45034 Fiskebaeckskil (Sweden)], E-mail: bodil.hernroth@marecol.gu.se

    2008-08-29

    Manganese (Mn) is a naturally abundant metal in marine sediments where it mainly occurs as MnO{sub 2}. During hypoxic conditions it is converted into a bioavailable state, Mn{sup 2+}, and can reach levels that previously have shown effects on immune competent cells of the crustacean, Nephrops norvegicus. Here we investigated if Mn also affects circulating coelomocytes and their renewal in the common sea star, Asterias rubens, when exposed to concentrations of Mn that can be found in nature. When the sea stars were exposed to Mn it accumulated in the coelomic fluid and the number of circulating coelomocytes, in contrast to what was recorded in Nephrops, increased significantly. By using the substitute nucleotide, 5-bromo-2'-deoxyuridine, BrdU, for tracing cell division and by recording mitotic index by nuclei staining, we found that Mn induced proliferation of cells from a putative haematopoietic tissue, the coelomic epithelium. In addition, the haematopoietic tissue and coelomocytes showed stress response in terms of changes in HSP70 levels and protein carbonyls, as judged by immunohistochemistry and Western blotting. Measurement of dehydrogenase activity, using MTS/PMS, revealed that Mn showed cytotoxic properties. We also found that the phagocytotic capacity of coelomocytes was significantly inhibited by Mn. It was concluded that the exposure of A. rubens to Mn induced renewal of coelomocytes and impaired their immune response.

  18. Manganese effects on haematopoietic cells and circulating coelomocytes of Asterias rubens (Linnaeus)

    International Nuclear Information System (INIS)

    Oweson, Carolina; Skoeld, Helen; Pinsino, Annalisa; Matranga, Valeria; Hernroth, Bodil

    2008-01-01

    Manganese (Mn) is a naturally abundant metal in marine sediments where it mainly occurs as MnO 2 . During hypoxic conditions it is converted into a bioavailable state, Mn 2+ , and can reach levels that previously have shown effects on immune competent cells of the crustacean, Nephrops norvegicus. Here we investigated if Mn also affects circulating coelomocytes and their renewal in the common sea star, Asterias rubens, when exposed to concentrations of Mn that can be found in nature. When the sea stars were exposed to Mn it accumulated in the coelomic fluid and the number of circulating coelomocytes, in contrast to what was recorded in Nephrops, increased significantly. By using the substitute nucleotide, 5-bromo-2'-deoxyuridine, BrdU, for tracing cell division and by recording mitotic index by nuclei staining, we found that Mn induced proliferation of cells from a putative haematopoietic tissue, the coelomic epithelium. In addition, the haematopoietic tissue and coelomocytes showed stress response in terms of changes in HSP70 levels and protein carbonyls, as judged by immunohistochemistry and Western blotting. Measurement of dehydrogenase activity, using MTS/PMS, revealed that Mn showed cytotoxic properties. We also found that the phagocytotic capacity of coelomocytes was significantly inhibited by Mn. It was concluded that the exposure of A. rubens to Mn induced renewal of coelomocytes and impaired their immune response

  19. Influence of elevated body temperature on circulating immunoglobulin-secreting cells

    DEFF Research Database (Denmark)

    Kappel, M; Barington, T; Gyhrs, A

    1995-01-01

    This work was designed to investigate the effect of in vivo hyperthermia in man on circulating immunoglobulin-secreting cells. Eight healthy male volunteers were immersed into a hot waterbath (WI) (water temperature 39.5 degrees C) for 2 h, whereby their body temperature rose to 39.5 degrees C....... On another occasion they served as their own controls, being immersed into thermoneutral water (water temperature 34.5 degrees C) for 2 h. Blood samples were drawn before immersion, at body temperatures of 38, 39 and 39.5 degrees C, as well as 2 h after WI when their body temperatures were normalized...

  20. Circulating tumor cells and miRNAs as prognostic markers in neuroendocrine neoplasms.

    Science.gov (United States)

    Zatelli, Maria Chiara; Grossrubatscher, Erika Maria; Guadagno, Elia; Sciammarella, Concetta; Faggiano, Antongiulio; Colao, Annamaria

    2017-06-01

    The prognosis of neuroendocrine neoplasms (NENs) is widely variable and has been shown to associate with several tissue- and blood-based biomarkers in different settings. The identification of prognostic factors predicting NEN outcome is of paramount importance to select the best clinical management for these patients. Prognostic markers have been intensively investigated, also taking advantage of the most modern techniques, in the perspective of personalized medicine and appropriate resource utilization. This review summarizes the available data on the possible role of circulating tumor cells and microRNAs as prognostic markers in NENs. © 2017 Society for Endocrinology.

  1. Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

    Directory of Open Access Journals (Sweden)

    Brian J Kirby

    Full Text Available Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI microfluidic device that combines an anti-prostate specific membrane antigen (PSMA antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new

  2. Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

    Science.gov (United States)

    Adams, Daniel L; Stefansson, Steingrimur; Haudenschild, Christian; Martin, Stuart S; Charpentier, Monica; Chumsri, Saranya; Cristofanilli, Massimo; Tang, Cha-Mei; Alpaugh, R Katherine

    2015-02-01

    Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley

  3. Increased circulating progenitor cells in Alzheimer's disease patients with moderate to severe dementia: evidence for vascular repair and tissue regeneration?

    Science.gov (United States)

    Stellos, Konstantinos; Panagiota, Victoria; Sachsenmaier, Saskia; Trunk, Theresia; Straten, Guido; Leyhe, Thomas; Seizer, Peter; Geisler, Tobias; Gawaz, Meinrad; Laske, Christoph

    2010-01-01

    Cerebrovascular dysfunction is a common finding in patients with Alzheimer's disease (AD) and may contribute to cognitive decline. Abundant evidence suggests that vascular and neuronal repair mechanisms are mediated by circulating progenitor cells in vivo. Whether CD34+ and, specifically, CD34+/CD133+ progenitor cells are involved in the pathophysiology of AD is poorly understood so far. In the present study, peripheral blood concentrations of circulating CD34+/CD133+ and CD34+ progenitor cells were measured in 45 AD patients and in 30 healthy elderly controls by flow cytometry. The severity of dementia was assessed by Mini-Mental Status Examination and Clinical Dementia Rating scale. AD patients were stratified into two groups showing mild (n=17) and moderate to severe (n= 28) dementia. In the present study, AD patients with moderate to severe dementia, but not those with mild dementia, showed significantly increased circulating CD34+/CD133+ and CD34+ progenitor cells compared to healthy elderly controls independent of cardiovascular risk factors and medication. In addition, the number of circulating CD34+/CD133+ progenitor cells in AD patients was significantly inversely correlated with cognitive function, age, and plasma levels of SDF-1, the most potent chemokine for progenitor cells. Our findings suggest a stage-dependent upregulation of circulating CD34+/CD133+ and CD34+ progenitor cells in AD patients, which could take part in tissue healing processes of the brain in AD.

  4. Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors.

    Science.gov (United States)

    Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Kajimura, Junko; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-01-01

    It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

  5. The Level of Circulating Endothelial Progenitor Cell Is Associated with Cerebral Vasoreactivity: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    2015-01-01

    Full Text Available Endothelial progenitor cell is known to be able to repair injured vessels. We assessed the hypothesis that endothelial progenitor cell also modulates cerebral endothelial function in healthy status. We used transcranial color-coded sonography to measure middle cerebral arterial vasoreactivity to CO2 (breath-holding index in healthy subjects and observed its relationship with the number of circulating CD34CD133+ cells. To detect significant correlations between each characteristic and breath-holding index of middle cerebral artery, we used univariate and multivariate regression analyses. 22 young healthy subjects were included in the present study (6 men, 16 women; mean age: 28.45 ± 3.98 years, range: 22–34 years. The mean breath-holding index and CD45lowCD34+CD133+ cells number were 0.95 ± 0.48% and 0.52 ± 0.26, respectively. The level of CD34CD133+ cells was independently associated with middle cerebral artery’s vasoreactivity (r=0.439,P=0.04. Our results suggest that endothelial progenitor cell also modulates healthy cerebral vessels’ endothelial function. This ability of endothelial progenitor cell could be potentially applied therapeutically and for prevention in conditions with cerebral endothelial dysfunction and cerebral ischemia.

  6. Cultured circulating tumor cells and their derived xenografts for personalized oncology

    Directory of Open Access Journals (Sweden)

    Ruoxiang Wang

    2016-10-01

    Full Text Available Recent cancer research has demonstrated the existence of circulating tumor cells (CTCs in cancer patient's blood. Once identified, CTC biomarkers will be invaluable tools for clinical diagnosis, prognosis and treatment. In this review, we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample, to derive enough CTCs for accurate and reproducible characterization of the biophysical, biochemical, gene expressional and behavioral properties of the harvested cells. Because of tumor cell heterogeneity, it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis. By analyzing critical steps and technical issues in ex vivo CTC culture, we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells, relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.

  7. Circulating hematopoietic stem cell count is a valuable predictor of prematurity complications in preterm newborns

    Directory of Open Access Journals (Sweden)

    Kotowski Maciej

    2012-09-01

    Full Text Available Abstract Background The frequency of preterm labour has risen over the last few years. Hence, there is growing interest in the identification of markers that may facilitate prediction and prevention of premature birth complications. Here, we studied the association of the number of circulating stem cell populations with the incidence of complications typical of prematurity. Methods The study groups consisted of 90 preterm (23–36 weeks of gestational age and 52 full-term (37–41 weeks infants. Non-hematopoietic stem cells (non-HSCs; CD45-lin-CD184+, enriched in very small embryonic-like stem cells (VSELs, expressing pluripotent (Oct-4, Nanog, early neural (β-III-tubulin, and oligodendrocyte lineage (Olig-1 genes as well as hematopoietic stem cells (HSCs; CD45+lin-CD184+, and circulating stem/progenitor cells (CSPCs; CD133+CD34+; CD133-CD34+ in association with characteristics of prematurity and preterm morbidity were analyzed in cord blood (CB and peripheral blood (PB until the sixth week after delivery. Phenotype analysis was performed using flow cytometry methods. Clonogenic assays suitable for detection of human hematopoietic progenitor cells were also applied. The quantitative parameters were compared between groups by the Mann–Whitney test and between time points by the Friedman test. Fisher’s exact test was used for qualitative variables. Results We found that the number of CB non-HSCs/VSELs is inversely associated with the birth weight of preterm infants. More notably, a high number of CB HSCs is strongly associated with a lower risk of prematurity complications including intraventricular hemorrhage, respiratory distress syndrome, infections, and anemia. The number of HSCs remains stable for the first six weeks of postnatal life. Besides, the number of CSPCs in CB is significantly higher in preterm infants than in full-term neonates (p  Conclusion We conclude that CB HSCs are markedly associated with the development of premature

  8. Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

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    Baumann Gert

    2010-05-01

    Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

  9. Residual stresses and strength of multilayer tape cast solid oxide fuel and electrolysis half-cells

    DEFF Research Database (Denmark)

    Charlas, Benoit; Frandsen, Henrik Lund; Brodersen, Karen

    2015-01-01

    -cells with the electrolyte on the compressive side corresponds to the strength of the support. With the loading in the other direction (electrolyte on the tensile side), the origin of the failure is in a different layer for MTC3 (fuel electrode) and for MTC4 (barrier layer). In order to decrease the tensile residual...... coefficient (TEC) mismatch between the layers, cumulated from high temperature, induces significant residual stresses in the half-cells. Furthermore, it has been observed that MTC half-cells with 4 layers (MTC4: support, fuel electrode, electrolyte and barrier layer) are sometimes more fragile to handle than...

  10. Quantitative determination of circulating endothelial cells in persons with low dose radioactivity exposure

    International Nuclear Information System (INIS)

    Al-Massarani, Gh.; Najjar, F.

    2013-04-01

    The aim of this study was to determine the risk of occupational exposure to low doses of ionizing radiation on the endothelium detachment through the quantification of circulating endothelial cells (CEC) in the peripheral blood of 63 workers in the Atomic Energy Commission in Syria (AECS) using a Magnetic Immuno-separation technique (IMS) and compare the results with 28 healthy (controls) is not exposed during their careers for any type of ionizing radiation. Our study showed for the first time the significantly increasing in the circulating endothelial cells count (P <0.0001) when employees are exposed to low doses of radiation less than 50 mSv. This result with previous studies about the late effects of radiation, assuming the existence of impact late radiation exposure on the cohesion of the endothelium, despite the lack of correlation with radiation dose measured during the past four years of work in AECS (between 2006-2010). This is due to several reasons, including the small sample size and lack of commitment by some workers develop individual control films during some periods of their work. The prospective studies for such workers can allow us to know if the rise in the number of CEC will be considered an early indicator for the risk of a cardiovascular disease when workers exposed to low-doses of ionizing radiation( tens of millisievert) that up to date are considered harmless (author).

  11. Effect of D-003 on intimal thickening and circulating endothelial cells in rabbit cuffed carotid artery.

    Science.gov (United States)

    Noa, Miriam; Mendoza, Sarahí; Más, Rosa

    2005-01-01

    D-003 is a mixture of very-high-molecular-weight aliphatic primary acids purified from sugar-cane (Saccharum officinarum L.) wax, in which octacosanoic acid is the most abundant component. Previous experimental studies have shown that D-003 not only shows cholesterol-lowering and anti-platelet effects, but also reduces thromboxane B2 and increases prostacyclin levels. It acts by inhibiting cholesterol biosynthesis. The positioning of a non-occlusive silicone collar around the rabbit carotid artery results in the formation of a neointima. Collars were placed around the left carotid for 15 days. The contralateral artery was sham-operated. We included three experimental groups: A control group received vehicle, and two others received D-003 at 5 and 25 mg/kg until sacrificed. Samples of arteries were examined by light microscopy. To evaluate intimal thickening the cross-sectional areas of intima and media were measured. Neointima was significantly reduced in D-003-treated animals compared with controls. Furthermore, the circulating endothelial cell has been studied in this experimental model with endothelium damage. The results demonstrate the protective effect of D-003 on vascular endothelium of the studied rabbits. It is concluded that the protective effect of D-003 against neointima formation and circulating endothelial cells in this experimental model could represent potential beneficial pleiotropic effects in the anti-atherogenic profile of this substance, beyond its cholesterol-lowering and anti-platelet effects independently demonstrated.

  12. Gene expression of circulating tumour cells and its correlation with tumour stage in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Bölke E

    2009-09-01

    Full Text Available Abstract Background Breast cancer (BC represents one of the leading causes of cancer related deaths worldwide. New tools for diagnostic staging and therapeutic monitoring are needed to improve individualized therapies and improve clinical outcome. The analyses of circulating tumour cells may provide important prognostic information in the clinical setting. Materials and methods Circulating tumour cells (CTC of 63 BC patients were isolated from peripheral blood (PB through immunomagnetic separation. Subsequently, RT-PCR or mPCR for the genes ga733.2, muc-1, c-erbB2, mgb-1, spdef and c-erbB2 were performed. Subsequently, expression data were correlated with the tumour stages. Fourteen healthy individuals served as controls. Results Significant correlations with tumour stages were found in single gene analyses of ga733.2, muc-1 and in multi-gene analyses of ga733.2/muc-1/mgb1/spdef. Furthermore, a significant correlation of Ca 15-3 and all studied genes was also observed. Conclusion Herein, we demonstrated a positive correlation of a gene signature consisting of ga733.2, muc-1, mgb1 and spdef and advanced stages of BC. Moreover, all studied genes and gene patterns revealed a significant correlation with Ca 15-3 positive cases.

  13. Residual β-cell function and the insulin-like growth factor system in Danish children and adolescents with type 1 diabetes

    DEFF Research Database (Denmark)

    Sorensen, Jesper S; Birkebaek, Niels H; Bjerre, Mette

    2015-01-01

    CONTEXT: C-peptide-positive adults with type 1 diabetes (T1D) have higher circulating total and free IGF-1 and lower IGF binding protein 1 (IGFBP-1) than C-peptide-negative patients. Whether this is also the case in children remains unknown. OBJECTIVE: The objective of the study was to examine...... the IGF system in children/adolescents with and without residual β-cell function (RBF). DESIGN AND PATIENTS: This was a cross-sectional study containing 136 prepubertal (hereof 15 RBF positive) and 206 pubertal (hereof 42 RBF positive) children/adolescents with T1D for 3-6 years as well as 40 prepubertal...

  14. Velocity effect on aptamer-based circulating tumor cell isolation in microfluidic devices.

    Science.gov (United States)

    Wan, Yuan; Tan, Jifu; Asghar, Waseem; Kim, Young-tae; Liu, Yaling; Iqbal, Samir M

    2011-12-01

    The isolation and detection of rare circulating tumor cells (CTCs) has been one of the focuses of intense research recently. In a microfluidic device, a number of factors can influence the enrichment capability of surface-bound probe molecules. This article analyzes the important factor of flow velocity in a microfluidic channel. The competition of surface-grafted anti-EGFR aptamers to bind the overexpressed EGFR on cell membranes against the drag force from the fluid flow is an important efficiency determining factor. The flow rate variations are applied both in experiments and in simulation models to study their effects on CTC capture efficiency. A mixture of mononuclear cells and human Glioblastoma cells is used to isolate cancer cells from the cellular flow. The results show interdependence between the adhesion probability, isolation efficiency, and flow rate. This work can help in designing flow-through lab-on-chip devices that use surface-bound probe affinities against overexpressed biomarkers for cell isolation. This work demonstrates that microfluidic based approaches have strong potential applications in CTC detection and isolation. © 2011 American Chemical Society

  15. Integrated Multifunctional Electrochemistry Microchip for Highly Efficient Capture, Release, Lysis, and Analysis of Circulating Tumor Cells.

    Science.gov (United States)

    Yan, Shuangqian; Chen, Peng; Zeng, Xuemei; Zhang, Xian; Li, Yiwei; Xia, Yun; Wang, Jie; Dai, Xiaofang; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

    2017-11-21

    The circulating tumor cells (CTCs) in the blood allow the noninvasive analysis of metastatic mechanisms, cancer diagnosis, prognosis, disease monitoring, and precise therapy through "liquid biopsies". However, there is no integrated and robust multifunctional microchip, which not only could highly efficient capture CTCs, but also fast release and lyse cells on one single chip without using other biochemical agents for downstream biomedical analysis. In this work, we integrated the three functions in one electrochemical microchip (echip) by intentionally designing a cactus-like, topologically structured conductive array consisted of a PDMS micropillar-array core and an electroconductive gold coating layer with hierarchical structure. The echip presented a capture efficiency of 85-100% for different cell lines in both buffer solution and whole blood. Moreover, the validity of the echip was further evaluated by using non-small-cell lung cancer patient samples. The electrochemical released cells or lysed-cell solutions could be obtained within 10 min and have been successfully used for mutant detection by DNA sequencing or RT-PCR. The fast release at a relative low voltage (-1.2 V) was originating from an electrochemical cleavage of the Au-S bonds that immobilized antibody on the chip. The electrochemical lysis took place at a high voltage (20 V) with an admirable performance. Thus, the highly integrated multifunctional echip was well demonstrated and promised a significant application in the clinical field.

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

    Science.gov (United States)

    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.

  17. Multispectral Imaging Analysis of Circulating Tumor Cells in Negatively Enriched Peripheral Blood Samples.

    Science.gov (United States)

    Miller, Brandon; Lustberg, Maryam; Summers, Thomas A; Chalmers, Jeffrey J

    2017-01-01

    A variety of biomarkers are present on cells in peripheral blood of patients with a variety of disorders, including solid tumor malignancies. While rare, characterization of these cells for specific protein levels with the advanced technology proposed, will lead to future validation studies of blood samples as "liquid biopsies" for the evaluation of disease status and therapeutic response. While circulating tumor cells (CTCs) have been isolated in the blood samples of patients with solid tumors, the exact role of CTCs as clinically useful predictive markers is still debated. Current commercial technology has significant bias in that a positive selection technology is used that preassumes specific cell surface markers (such as EpCAM) are present on CTCs. However, CTCs with low EpCAM expression have been experimentally demonstrated to be more likely to be missed by this method. In contrast, this application uses a previously developed, technology that performs a purely negative enrichment methodology on peripheral blood, yielding highly enriched blood samples that contain CTCs as well as other, undefined cell types. The focus of this contribution is the use of multispectral imaging of epifluorescent, microscopic images of these enriched cells in order to help develop clinically relevant liquid biopsies from peripheral blood samples.

  18. Passive circulating cell sorting by deformability using a microfluidic gradual filter.

    Science.gov (United States)

    Preira, P; Grandné, V; Forel, J-M; Gabriele, S; Camara, M; Theodoly, O

    2013-01-07

    The deformability of circulating leukocytes plays an important role in the physiopathology of several diseases like sepsis or acute respiratory distress syndrome (ARDS). We present here a microfluidic method for the passive testing, sorting and separating of non-adherent cell populations by deformability. It consists of microfluidic sieves in series with pore sizes decreasing from the upstream to the downstream. The method capabilities are demonstrated with monocytic cell lines (THP-1) treated by Jasplakinolide (a stabilizer of polymerized actin), LatrunculinA (an inhibitor of actin polymerization), and with the plasma of patients suffering from ARDS. Simple sample injection with standard syringes and pumps makes the method readily adapted for experimentation in hospitals.

  19. Dynamic Changes in Numbers and Properties of Circulating Tumor Cells and Their Potential Applications

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Ju-Yu [Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei 11221, Taiwan (China); Yang, Chih-Yung [Department of Education and Research, Taipei City Hospital, Taipei 10629, Taiwan (China); Liang, Shu-Ching [Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei 11221, Taiwan (China); Liu, Ren-Shyan [Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, Taipei 11529, Taiwan (China); Biomedical Imaging Research Center, Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan (China); National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, 11217, Taiwan (China); Jiang, Jeng-Kai, E-mail: jkjiang@vghtpe.gov.tw [Division of Colon & Rectal Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei 11217, Taiwan (China); Lin, Chi-Hung, E-mail: jkjiang@vghtpe.gov.tw [Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei 11221, Taiwan (China); VGH Yang-Ming Genome Research Center, Taipei 11221, Taiwan (China)

    2014-12-16

    Circulating tumor cells (CTCs) can be detected in the blood of different types of early or advanced cancer using immunology-based assays or nucleic acid methods. The detection and quantification of CTCs has significant clinical utility in the prognosis of metastatic breast, prostate, and colorectal cancers. CTCs are a heterogeneous population of cells and often different from those of their respective primary tumor. Understanding the biology of CTCs may provide useful predictive information for the selection of the most appropriate treatment. Therefore, CTC detection and characterization could become a valuable tool to refine prognosis and serve as a “real-time biopsy” and has the potential to guide precision cancer therapies, monitor cancer treatment, and investigate the process of metastasis.

  20. Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury

    DEFF Research Database (Denmark)

    Hagensen, Mette; Raarup, Merete Krog; Mortensen, Martin Bødtker

    2012-01-01

    into endothelial cells (ECs). We tested this theory in a murine arterial injury model using carotid artery transplants and fluorescent reporter mice. METHODS AND RESULTS: Wire-injured carotid artery segments from wild-type mice were transplanted into TIE2-GFP transgenic mice expressing green fluorescent protein...... (GFP) in ECs. We found that the endothelium regenerated with GFP(+) ECs as a function of time, evolving from the anastomosis sites towards the centre of the transplant. A migration front of ECs at Day 7 was verified by scanning electron microscopy and by bright-field microscopy using recipient TIE2-lac......Z mice with endothelial β-galactosidase expression. These experiments indicated migration of flanking ECs rather than homing of circulating cells as the underlying mechanism. To confirm this, we interposed non-injured wild-type carotid artery segments between the denuded transplant and the TIE2-GFP...

  1. Circulating endothelial progenitor cells: a new approach to anti-aging medicine?

    Directory of Open Access Journals (Sweden)

    Patel Amit N

    2009-12-01

    Full Text Available Abstract Endothelial dysfunction is associated with major causes of morbidity and mortality, as well as numerous age-related conditions. The possibility of preserving or even rejuvenating endothelial function offers a potent means of preventing/treating some of the most fearful aspects of aging such as loss of mental, cardiovascular, and sexual function. Endothelial precursor cells (EPC provide a continual source of replenishment for damaged or senescent blood vessels. In this review we discuss the biological relevance of circulating EPC in a variety of pathologies in order to build the case that these cells act as an endogenous mechanism of regeneration. Factors controlling EPC mobilization, migration, and function, as well as therapeutic interventions based on mobilization of EPC will be reviewed. We conclude by discussing several clinically-relevant approaches to EPC mobilization and provide preliminary data on a food supplement, Stem-Kine, which enhanced EPC mobilization in human subjects.

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

    Science.gov (United States)

    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.

  3. Next-generation sequencing-based detection of circulating tumour DNA After allogeneic stem cell transplantation for lymphoma.

    Science.gov (United States)

    Herrera, Alex F; Kim, Haesook T; Kong, Katherine A; Faham, Malek; Sun, Heather; Sohani, Aliyah R; Alyea, Edwin P; Carlton, Victoria E; Chen, Yi-Bin; Cutler, Corey S; Ho, Vincent T; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J; Soiffer, Robert J; Antin, Joseph H; Armand, Philippe

    2016-12-01

    Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3·7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% vs. 84% in ctDNA-negative patients, P = 0·033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3·9, P = 0·003) and increased risk of relapse/progression (Hazard ratio 10·8, P = 0·0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. © 2016 John Wiley & Sons Ltd.

  4. The effects of phototherapy on the numbers of circulating natural killer cells and T lymphocytes in psoriasis.

    LENUS (Irish Health Repository)

    Tobin, A M

    2009-04-01

    The innate immune system is believed to be important in the pathogenesis of psoriasis and natural killer (NK) have been found in increased numbers in psoriatic plaques. Alterations in the numbers of NK cells in peripheral blood have been reported. We investigated the effect of phototherapy on levels of peripheral NK cells and lymphocytes in patients with psoriasis. In nine patients whom we followed before, during and after narrowband ultraviolet B (UVB) treatment there were no differences in the numbers of circulating lymphocytes, lymphocyte subsets or cells expressing NK markers and controls. Treatment with narrowband UVB did, however, significantly lower circulating CD4 counts which gradually recovered posttreatment.

  5. Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

    Science.gov (United States)

    Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

    2018-01-30

    Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

  6. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    Science.gov (United States)

    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.

  7. Diurnal Variations of Human Circulating Cell-Free Micro-RNA.

    Directory of Open Access Journals (Sweden)

    Niels H H Heegaard

    Full Text Available A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks. The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79 of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks.

  8. Pre-Clinical Evaluation of Biopolymer Delivered Circulating Angiogenic Cells in Hibernating Myocardium

    Science.gov (United States)

    Giordano, Celine

    Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization strategy for patients with hibernating myocardium, a common clinical condition. We used a clinically relevant swine model of hibernating myocardium to examine the benefits of biopolymer-supported delivery of circulating angiogenic cells (CACs) in this context. Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, positron emission tomography measures of myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the affected region (both pstress MBF and MFR were increased only in the cells+matrix group (panimals (p=0.02) compared to controls. Similar results were found using microsphere-measured MBF. Wall motion abnormalities and ejection fraction improved only in the cells+matrix group. This preclinical swine model demonstrated ischemia and hibernation, which was improved by the combined delivery of CACs and a collagen-based matrix. To our knowledge, this is the first demonstration of the mechanisms and effects of combining progenitor cells and biopolymers in the setting of myocardial hibernation, a common clinical condition in patients with advanced coronary artery disease.

  9. Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques.

    Science.gov (United States)

    Campos, María; Prior, Celia; Warleta, Fernando; Zudaire, Isabel; Ruíz-Mora, Jesús; Catena, Raúl; Calvo, Alfonso; Gaforio, José J

    2008-07-01

    The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies.

  10. Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Stephen D. Mikolajczyk

    2011-01-01

    Full Text Available Enrichment of rare circulating tumor cells (CTCs in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM, with detection using cytokeratin (CK antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE, a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs, while all samples contained additional CE-positive cells (range 1–41; median = 11; =.02. Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

  11. Continuous enrichment of circulating tumor cells using a microfluidic lateral flow filtration chip.

    Science.gov (United States)

    Lee, Sung-Woo; Hyun, Kyung-A; Kim, Seung-Il; Kang, Ji-Yoon; Jung, Hyo-Il

    2015-01-16

    The isolation and characterization of circulating tumor cells (CTC) is of great importance in cancer diagnosis and prognosis. Highly sensitive detection of CTCs can be very difficult because they are extremely rare (i.e., 1-5 CTCs per 10(9) erythrocytes) in blood. Recently, various devices have been developed that exploit biochemical (affinity-based) and physical (size or density) methods. Antibody-based isolation has its own limitations, as the expression level of the epitopes for an antibody varies due to the heterogeneity of cancer cells. Harsh conditions associated with physical methods can cause the deformation and damage of CTCs during the isolation process. Here, we propose a microfluidic lateral flow filtration (μ-LaFF) chip in which lateral flow was combined with vertical flow into the filter to capture the CTCs gently. The CTCs experienced weak shear flow owing to the lateral flow and traveled alongside the filter channel until finally being captured. The vertical flow in the filter held the captured cells tightly and served as an exit for uncaptured hematological cells (white and red blood cells). From our μ-LaFF chip we obtained a high capture efficiency (95%) and purity (99%), minimizing any damage to the CTCs. Our μ-LaFF technology is expected to be useful in the diagnosis and prognosis of various cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Residual stresses in a co-sintered SOC half-cell during post-sintering cooling

    DEFF Research Database (Denmark)

    Charlas, Benoit; Chatzichristodoulou, Christodoulos; Brodersen, Karen

    2014-01-01

    on the loading conditions, the additional stress needed to break the cells can indeed be smaller due to the initial thermo-mechanical stress state. The residual stresses can for a bilayer cell be approximated by estimating the temperature at which elastic stresses start to build up during the cooling, i.......e. the reference temperature (Tref) or the strain difference based on the curvature. This approximation gives good results for bilayers with a defined cooling temperature profile, where the curvature of the bilayer defines a unique balance between the two unknown residual stress states in the two layers...... simulation taking into account the elastic response and creep of each layer. The model is validated by measuring the curvature and residual stresses of multi-layer cells....

  13. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    Science.gov (United States)

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  14. Persistent Reduction of Circulating Myeloid Calcifying Cells in Acromegaly: Relevance to the Bone-Vascular Axis.

    Science.gov (United States)

    Fadini, Gian Paolo; Dassie, Francesca; Cappellari, Roberta; Persano, Mariasara; Vigili de Kreutzenberg, Saula; Martini, Chiara; Parolin, Matteo; Avogaro, Angelo; Vettor, Roberto; Maffei, Pietro

    2017-06-01

    Acromegaly is a systemic disease characterized by persistent bone pathology and excess cardiovascular mortality. Despite multiple concomitant risk factors, atherosclerosis does not seem to be accelerated in acromegaly. To compare the levels of circulating myeloid calcifying cells (MCCs), which promote ectopic calcification and inhibit angiogenesis, in individuals with and without acromegaly. Cross-sectional case-control study. Tertiary ambulatory referral endocrinology center. 44 acromegalic patients (25 active; 19 inactive), 44 control subjects matched by age, sex, risk factors, and medications, and 8 patients cured of acromegaly. MCCs were measured using flow cytometry based on the expression of osteocalcin (OC) and bone alkaline phosphatase (BAP) on monocytes and circulating CD34+ stem cells. Differences in MCCs between patients and controls. OC+BAP+ MCCs were severely reduced in acromegalic compared with control patients (0.17% ± 0.02% vs 1.00% ± 0.24%; P < 0.001), as were the total OC+ and BAP+ monocytic cells. Patients with inactive acromegaly and those cured of acromegaly displayed persistently reduced levels of MCCs. In the controls, but not acromegalic patients, MCCs were increased in the presence of diabetes or cardiovascular disease. A direct correlation was noted between MCCs and parathyroid hormone (r = 0.61; P < 0.0001), supporting a link between bone biology and MCCs. In patients with acromegaly, the levels of MCCs are reduced and remain low, even years after a complete cure. This finding might be related to low atherosclerotic calcification and the persistence of bone pathology after acromegaly remission or cure. Copyright © 2017 Endocrine Society

  15. Circulating Endothelial Progenitor Cells in Type 1 Diabetic Patients: Relation with Patients’ Age and Disease Duration

    Directory of Open Access Journals (Sweden)

    Adolfo Arcangeli

    2017-10-01

    Full Text Available ObjectivesCirculating endothelial progenitor cells (cEPCs have been reported to be dysfunctional in diabetes mellitus (DM patients, accounting for the vascular damage and the ensuing high risk for cardiovascular disease (CVD characteristic of this disease. The aim of the present study was to evaluate the number of circulating cEPCs in type 1 DM (T1DM patients, without clinical vascular damage, of different ages and with different disease duration.MethodsAn observational, clinical-based prospective study was performed on T1DM patients enrolled in two clinical centers. cEPCs were determined by flow cytometry, determining the number of CD34/CD133/VEGFR2-positive cells within peripheral blood mononuclear cells (PBMCs.ResultsThe number of cEPCs was lower in adult T1DM patients, whilst higher in childhood/young patients, compared to controls of the same age range. When patients were grouped into two age groups (≥ or <20 years (and categorized on the basis of the duration of the disease, the number of cEPCs in young (<20 years patients was higher compared with older subjects, regardless of disease duration. A subset of patients with very high cEPCs was identified in the <20 years group.ConclusionThere is an association between the number of cEPCs and patients’ age: childhood/young T1DM patients have significantly higher levels of cEPCs, respect to adult T1DM patients. Such difference is maintained also when the disease lasts for more than 10 years. The very high levels of cEPCs, identified in a subset of childhood/young patients, might protect vessels against endothelial dysfunction and damage. Such protection would be less operative in older subjects, endowed with lower cEPC numbers, in which complications are known to develop more easily.

  16. Increased circulating fibrocytes are associated with higher reticulocyte percent in children with sickle cell anemia.

    Science.gov (United States)

    Karafin, Matthew S; Dogra, Shibani; Rodeghier, Mark; Burdick, Marie; Mehrad, Borna; Rose, C Edward; Strieter, Robert M; DeBaun, Michael R; Strunk, Robert C; Field, Joshua J

    2016-03-01

    Interstitial lung disease is common in patients with sickle cell anemia (SCA). Fibrocytes are circulating cells implicated in the pathogenesis of pulmonary fibrosis and airway remodeling in asthma. In this study, we tested the hypotheses that fibrocyte levels are: (1) increased in children with SCA compared to healthy controls, and (2) associated with pulmonary disease. Cross-sectional cohort study of children with SCA who participated in the Sleep Asthma Cohort Study. Fibrocyte levels were obtained from 45 children with SCA and 24 controls. Mean age of SCA cases was 14 years and 53% were female. In children with SCA, levels of circulating fibrocytes were greater than controls (P < 0.01). The fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on the majority of cells and CCR2 and CCR7 expressed on a smaller subset. Almost half of fibrocytes demonstrated α-smooth muscle actin activation. Increased fibrocyte levels were associated with a higher reticulocyte count (P = 0.03) and older age (P = 0.048) in children with SCA. However, children with increased levels of fibrocytes were not more likely to have asthma or lower percent predicted forced expiratory volume in 1 sec/forced vital capacity (FEV1 /FVC) or FEV1 than those with lower fibrocyte levels. Higher levels of fibrocytes in children with SCA compared to controls may be due to hemolysis. Longitudinal studies may be able to better assess the relationship between fibrocyte level and pulmonary dysfunction. © 2015 Wiley Periodicals, Inc.

  17. Perspective on Circulating Tumor Cell Clusters: Why It Takes a Village to Metastasize.

    Science.gov (United States)

    Giuliano, Mario; Shaikh, Anum; Lo, Hin Ching; Arpino, Grazia; De Placido, Sabino; Zhang, Xiang H; Cristofanilli, Massimo; Schiff, Rachel; Trivedi, Meghana V

    2018-02-15

    Circulating tumor cell (CTC) clusters may represent one of the key mechanisms initiating the metastasis process. However, the series of pathophysiologic events by which CTC clusters originate, enter the circulation, and reach the distant sites remain to be identified. The cellular and molecular mechanisms that provide survival advantage for CTC clusters during the transit in the blood stream are also still largely unknown. Understanding the biology of CTC clusters is critical to assess this unified scheme employed by cancer and to device strategies to overcome key pathways responsible for their improved metastatic potential. CTC clusters remain an underdeveloped area of research begging the attention of multidisciplinary cancer research teams. Here, we provide insight on existing preclinical evidence on the potential mechanisms leading to CTC cluster formation and dissemination and on processes that may offer survival advantage. We also offer our perspective on future directions to delineate the role of CTC clusters in metastatic cascade and discuss their clinical significance. Cancer Res; 78(4); 845-52. ©2018 AACR . ©2018 American Association for Cancer Research.

  18. Obesity suppresses circulating level and function of endothelial progenitor cells and heart function

    Directory of Open Access Journals (Sweden)

    Tsai Tzu-Hsien

    2012-07-01

    Full Text Available Abstract Background and aim This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs and left ventricular ejection fraction (LVEF. Methods High fat diet (45 Kcal% fat was given to 8-week-old C57BL/6 J mice (n = 8 for 20 weeks to induce obesity (group 1. Another age-matched group (n = 8 were fed with control diet for 20 weeks as controls (group 2. The animals were sacrificed at the end of 20 weeks after obesity induction. Results By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p Conclusions Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

  19. MRA of the intracranial circulation in asymptomatic patients with sickle cell disease

    International Nuclear Information System (INIS)

    Gillams, A.R.; McMahon, L.; Weinberg, G.; Carter, A.P.

    1998-01-01

    Background. MR angiography (MRA) provides a mechanism for non-invasively studying blood flow, thus providing a new opportunity to study the intracranial circulation in asymptomatic sickle cell disease (SCD) patients. Although conventional angiography is the gold standard for the depiction of vascular anatomy, this is too invasive for an asymptomatic population. Objective. To establish the range of appearances in asymptomatic SCD patients and to correlate brain MRI results (either sub-clinical abnormalities or normal brain parenchyma) with the MRA findings. Materials and methods. Brain MRI and MRA of the intracranial circulation was performed on 22 patients (13 male and 9 female, median age 7.5 years, range 1.3-20 years). Fourteen were homozygous SS and eight were SC. The median haematocrit at the time of MRI was 25.9 (range 13.8-33.3). Results. On MR imaging, four patients had infarcts in eight vascular territories (six anterior and two posterior). In 3/4 of anterior vascular territories with infarction, long (≥ 6 mm) segments of abnormal signal were seen at the internal carotid artery bifurcation with associated reduced distal flow. Short focal areas of abnormal signal were commonly seen where vessels branched, bifurcated or curved and were not associated with infarcts. These areas probably represent turbulence-related dephasing secondary to high velocity flow found in SCD. Conclusion. Long segments (≥ 6 mm) of abnormal signal with reduced distal flow correlated with sub-clinical infarction. (orig.)

  20. Cutaneous histopathology of Sézary syndrome: a study of 41 cases with a proven circulating T-cell clone.

    Science.gov (United States)

    Trotter, M J; Whittaker, S J; Orchard, G E; Smith, N P

    1997-05-01

    Sezary syndrome is an uncommon variant of cutaneous T-cell lymphoma (CTCL) characterized by erythroderma, pruritus, adenopathy, and circulating atypical T-lymphocytes with cerebriform nuclei. The definition of Sezary syndrome can be further refined by including only patients with a circulating peripheral blood population of clonal T-cells. We have evaluated 79 skin biopsies from such a group of 41 erythrodermic patients with circulating Sezary cells and a clonal population of T-cells detected by T-cell receptor-beta gene rearrangement on Southern analysis of peripheral blood mononuclear cells. Histopathologic features consistent with chronic dermatitis were observed in 26/79 (33%) skin biopsy specimens, emphasizing that a non-specific histologic appearance is common. Evidence of CTCL was lacking in 11/41 patients on biopsy of their erythrodermic skin. The survival of these patients was not significantly different from 30/41 patients in whom skin biopsies revealed changes diagnostic of CTCL, such as a dermal lymphocytic band with atypical lymphocytes (18/79, 23%) or a mycosis fungoides-like infiltrate (30/79, 38%). This study confirms that non-specific cutaneous histopathologic findings are common in Sezary syndrome, even when a circulating T-cell clone is present. This stresses the need for peripheral blood genetic analysis and for multiple or repeat skin biopsies in erythrodermic patients when there is high clinical suspicion of CTCL.

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

    Science.gov (United States)

    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

    2015-06-01

    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

  2. Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

    Science.gov (United States)

    Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

    2008-11-01

    Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times

  3. Circulating endothelial progenitor cell numbers are not associated with donor organ age or allograft vasculopathy in cardiac transplant recipients.

    Science.gov (United States)

    Thomas, H E; Parry, G; Dark, J H; Arthur, H M; Keavney, B D

    2009-02-01

    Increasing age is associated with reduced numbers of circulating endothelial progenitor cells (EPCs). It is unclear whether this relates to depletion or impairment of bone marrow progenitors, or to deficient mobilization signals from aging tissues. In cardiac transplant patients, one previous study has reported an association between circulating EPCs and the risk of cardiac allograft vasculopathy (CAV). We investigated whether increased donor heart age, a strong risk factor for CAV, was associated with reduced circulating EPC numbers in a group of cardiac transplant recipients matched for factors which influence EPC numbers, but with maximally discordant donor heart ages. We identified 32 patient pairs, matched for factors known to influence EPC numbers, but who had discordant donor heart ages by at least 20 years. EPCs were quantified using flow cytometry for absolute counts of cells expressing all the combinations of CD45, CD34, CD133 and the kinase domain receptor (KDR). There were no significant differences in the numbers of circulating EPCs between patients with old or young donor heart age. There was no association between the presence of CAV and circulating EPC numbers. We suggest that the increased susceptibility to CAV of older donor hearts is not mediated via circulating EPCs. Our results are consistent with the theory that the normal age-related decline in EPC numbers relates to bone marrow aging rather than failure of target tissues to induce EPC mobilization.

  4. High levels of peripheral blood circulating plasma cells as a specific risk factor for progression of smoldering multiple myeloma.

    Science.gov (United States)

    Bianchi, G; Kyle, R A; Larson, D R; Witzig, T E; Kumar, S; Dispenzieri, A; Morice, W G; Rajkumar, S V

    2013-03-01

    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 >5 × 10(6)/l and/or >5% PCs per 100 cytoplasmic immunoglobulin (Ig)-positive 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 24%, respectively, P=0.001. Corresponding rates for progression within 3 years were 86% versus 34%, 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 2-3 years following diagnosis.

  5. A SYSTEM AND A DEVICE FOR ISOLATING CIRCULATING TUMOR CELLS FROM THE PERIPHERAL BLOOD IN VIVO

    Directory of Open Access Journals (Sweden)

    Michal Mego

    2015-08-01

    Full Text Available Circulating tumor cells (CTC play a crucial role in disseminating tumors and in the metastatic cascade. CTCs are found only in small numbers, and the limited amount of isolated CTCs makes it impossible to characterize them closely. This paper presents a proposal for a new system for isolating CTCs from the peripheral blood in vivo. The system enables CTCs to be isolated from the whole blood volume for further research and applications. The proposed system consists of magnetic nanoparticles covered by monoclonal antibodies against a common epithelial antigen, large supermagnets, which are used to control the position of the nanoparticles within the human body, and a special wire made of a magnetic core wrapped in a non-magnetic shell. The system could be used not only for isolating CTCs, but also for in vivo isolation of other rare cells from the peripheral blood, including hematopoietic and/or mesenchymal stem cells, with applications in regenerative medicine and/or in stem cell transplantation.

  6. Circulating cell-free DNA for non-invasive cancer management.

    Science.gov (United States)

    Stewart, Caitlin M; Tsui, Dana W Y

    2018-03-11

    Cell-free DNA (cfDNA) was first identified in human plasma in 1948 and is thought to be released from cells throughout the body into the circulatory system. In cancer, a portion of the cfDNA originates from tumour cells, referred to as circulating-tumour DNA (ctDNA), and can contain mutations corresponding to the patient's tumour, for instance specific TP53 alleles. Profiling of cfDNA has recently become an area of increasing clinical relevance in oncology, in particular due to advances in the sensitivity of molecular biology techniques and development of next generation sequencing technologies, as this allows tumour mutations to be identified and tracked non-invasively. This has opened up new possibilities for monitoring tumour evolution and acquisition of resistance, as well as for guiding treatment decisions when tumour biopsy tissue is insufficient or unavailable. In this review, we will discuss the biology of cell-free nucleic acids, methods of analysis, and the potential clinical uses of these techniques, as well as the on-going clinical development of ctDNA assays. Copyright © 2018. Published by Elsevier Inc.

  7. hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells.

    Science.gov (United States)

    Schanz, Andrea; Lukosz, Margarete; Hess, Alexandra P; Baston-Büst, Dunja M; Krüssel, Jan S; Heiss, Christian

    2015-08-01

    Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation. Copyright © 2015. Published by Elsevier Ireland Ltd.

  8. Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells.

    Science.gov (United States)

    Wong, Keith H K; Tessier, Shannon N; Miyamoto, David T; Miller, Kathleen L; Bookstaver, Lauren D; Carey, Thomas R; Stannard, Cleo J; Thapar, Vishal; Tai, Eric C; Vo, Kevin D; Emmons, Erin S; Pleskow, Haley M; Sandlin, Rebecca D; Sequist, Lecia V; Ting, David T; Haber, Daniel A; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet

    2017-11-23

    Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

  9. Carboxybetaine methacrylate-modified nylon surface for circulating tumor cell capture.

    Science.gov (United States)

    Wang, Huiyu; Yue, Guofeng; Dong, Chaoqun; Wu, Fenglei; Wei, Jia; Yang, Yang; Zou, Zhengyun; Wang, Lifeng; Qian, Xiaoping; Zhang, Tao; Liu, Baorui

    2014-03-26

    Conventional in vitro circulating tumor cell (CTC) detection methods are always limited by blood sample volume because of the requirement of a large amount of blood. The aim of this study was to overcome the limitation by designing and making an in vivo CTC capture device. In this study, we designed and prepared a kind of proper material to serve the purpose of intervention. A method employing 3-aminopropyltriethoxysilane (γ-APS) as the coupling reagent to graft carboxybetaine methacrylate (CBMA) and to immobilize an anti-epithelial cell adhesion molecular (EpCAM) antibody on Nylon was developed. The results of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy proved the successful graft of γ-APS and CBMA to Nylon. Furthermore, the predicted improvement in the biocompatibilities of our modified Nylon was confirmed by water contact angle measurement, bovine serum albumin adhesion, platelet adhesion, plasma recalcification time determination, and cytotoxicity tests. The tumor cells adhesion experiment revealed that Nylon with the antibody immobilized on it had an affinity for EpCAM positive tumor cells higher than that of pristine Nylon. Additionally, the capture ability of the CTCs was demonstrated in a nude mouse tumor model using the interventional device made of the modified Nylon wire. The positive results suggest that CBMA-grafted and anti-EpCAM antibody-immobilized Nylon is a promising new material for in vivo CTC capture devices.

  10. Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

    Directory of Open Access Journals (Sweden)

    Kui-Jin Kim

    Full Text Available Basal-like breast carcinomas (BLCs present with extratumoral lymphovascular invasion, are highly metastatic, presumably through a hematogenous route, have augmented expression of CD44 oncoprotein and relatively low levels of retinoblastoma (Rb tumor suppressor. However, the causal relation among these features is not clear. Here, we show that Rb acts as a key suppressor of multiple stages of metastatic progression. Firstly, Rb suppresses collective cell migration (CCM and CD44-dependent formation of F-actin positive protrusions in vitro and cell-cluster based lymphovascular invasion in vivo. Secondly, Rb inhibits the release of single cancer cells and cell clusters into the hematogenous circulation and subsequent metastatic growth in lungs. Finally, CD44 expression is required for collective motility and all subsequent stages of metastatic progression initiated by loss of Rb function. Altogether, our results suggest that Rb/CD44 pathway is a crucial regulator of CCM and metastatic progression of BLCs and a promising target for anti-BLCs therapy.

  11. A Two-Stage Microfluidic Device for the Isolation and Capture of Circulating Tumor Cells

    Science.gov (United States)

    Cook, Andrew; Belsare, Sayali; Giorgio, Todd; Mu, Richard

    2014-11-01

    Analysis of circulating tumor cells (CTCs) can be critical for studying how tumors grow and metastasize, in addition to personalizing treatment for cancer patients. CTCs are rare events in blood, making it difficult to remove CTCs from the blood stream. Two microfluidic devices have been developed to separate CTCs from blood. The first is a double spiral device that focuses cells into streams, the positions of which are determined by cell diameter. The second device uses ligand-coated magnetic nanoparticles that selectively attach to CTCs. The nanoparticles then pull CTCs out of solution using a magnetic field. These two devices will be combined into a single 2-stage microfluidic device that will capture CTCs more efficiently than either device on its own. The first stage depletes the number of blood cells in the sample by size-based separation. The second stage will magnetically remove CTCs from solution for study and culturing. Thus far, size-based separation has been achieved. Research will also focus on understanding the equations that govern fluid dynamics and magnetic fields in order to determine how the manipulation of microfluidic parameters, such as dimensions and flow rate, will affect integration and optimization of the 2-stage device. NSF-CREST: Center for Physics and Chemistry of Materials. HRD-0420516; Department of Defense, Peer Reviewed Medical Research Program Award W81XWH-13-1-0397.

  12. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Sun Jianxiang; Sun Weijian; Sui Jianli; Zhou Pingkun

    2008-01-01

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60 Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D 0 or SF 2 ). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  13. Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase-positive cells.

    Science.gov (United States)

    Takakura, Masahiro; Matsumoto, Takeo; Nakamura, Mitsuhiro; Mizumoto, Yasunari; Myojyo, Subaru; Yamazaki, Rena; Iwadare, Jyunpei; Bono, Yukiko; Orisaka, Shunsuke; Obata, Takeshi; Iizuka, Takashi; Kagami, Kyosuke; Nakayama, Kentaro; Hayakawa, Hideki; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Urata, Yasuo; Fujiwara, Toshiyoshi; Kyo, Satoru; Sasagawa, Toshiyuki; Fujiwara, Hiroshi

    2018-01-01

    Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  14. Maternal Body-Mass Index and Cord Blood Circulating Endothelial Colony-Forming Cells

    Science.gov (United States)

    Lin, Ruei-Zeng; Miranda, Maria L.; Vallejo-Vaz, Antonio J.; Stiefel, Pablo; Praena-Fernández, Juan M.; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M.; Villar, Jose; Melero-Martin, Juan M.

    2013-01-01

    Objective Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in non-pathological pregnancies. Study design We measured the level of ECFCs in the cord blood of neonates (n=27) born from non-obese healthy mothers with non-pathological pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. Results We observed variation in ECFC abundance among subjects and found a positive correlation between pre-pregnancy maternal BMI and ECFC content (r=0.51, P=0.007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m2 and BMI between 25–30 kg/m2, including the ability to form vascular networks in vivo. Conclusions This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathological pregnancies. Endothelial colony-forming cells (ECFCs) are a subset of progenitor cells that circulate in peripheral blood and can give rise to endothelial cells (1,2), contributing to the formation of new vasculature and the maintenance of vascular integrity (3–5). The mechanisms that regulate the abundance of these cells in vivo remain poorly understood. ECFCs are rare in adult peripheral blood (1,2,10). In contrast, there is an elevated number of these cells in fetal blood during the third trimester of pregnancy (11–13). Emerging evidence indicates that deleterious conditions during fetal life can impair ECFC content and function. For instance, offspring of diabetic mothers have been shown to have

  15. Androgen receptor expression on circulating tumor cells in metastatic breast cancer.

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    Takeo Fujii

    Full Text Available Androgen receptor (AR is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+ breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer.Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK, and biomarkers of interest (AR, estrogen receptor [ER], and HER2 on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms.Of 68 patients, 51 (75% had at least 1 CTC, and 49 of these 51 (96% had hormone-receptor-positive (HR+/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells.CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

  16. Detection and Clinical Significance of Circulating Tumor Cells in Colorectal Cancer--20 Years of Progress.

    Science.gov (United States)

    Hardingham, Jennifer E; Grover, Phulwinder; Winter, Marnie; Hewett, Peter J; Price, Timothy J; Thierry, Benjamin

    2015-10-27

    Circulating tumor cells (CTC) may be defined as tumor- or metastasis-derived cells that are present in the bloodstream. The CTC pool in colorectal cancer (CRC) patients may include not only epithelial tumor cells, but also tumor cells undergoing epithelial-mesenchymal transition (EMT) and tumor stem cells. A significant number of patients diagnosed with early stage CRC subsequently relapse with recurrent or metastatic disease despite undergoing "curative" resection of their primary tumor. This suggests that an occult metastatic disease process was already underway, with viable tumor cells being shed from the primary tumor site, at least some of which have proliferative and metastatic potential and the ability to survive in the bloodstream. Such tumor cells are considered to be responsible for disease relapse in these patients. Their detection in peripheral blood at the time of diagnosis or after resection of the primary tumor may identify those early-stage patients who are at risk of developing recurrent or metastatic disease and who would benefit from adjuvant therapy. CTC may also be a useful adjunct to radiological assessment of tumor response to therapy. Over the last 20 years many approaches have been developed for the isolation and characterization of CTC. However, none of these methods can be considered the gold standard for detection of the entire pool of CTC. Recently our group has developed novel unbiased inertial microfluidics to enrich for CTC, followed by identification of CTC by imaging flow cytometry. Here, we provide a review of progress on CTC detection and clinical significance over the last 20 years.

  17. Androgen receptor expression on circulating tumor cells in metastatic breast cancer

    Science.gov (United States)

    Fujii, Takeo; Reuben, James M.; Huo, Lei; Espinosa Fernandez, Jose Rodrigo; Gong, Yun; Krupa, Rachel; Suraneni, Mahipal V.; Graf, Ryon P.; Lee, Jerry; Greene, Stephanie; Rodriguez, Angel; Dugan, Lyndsey; Louw, Jessica; Lim, Bora; Barcenas, Carlos H.; Marx, Angela N.; Tripathy, Debu; Wang, Yipeng; Landers, Mark; Dittamore, Ryan

    2017-01-01

    Purpose Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer. Methods Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms. Results Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells. Conclusions CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their

  18. Kinetics of circulating endothelial progenitor cells in patients undergoing carotid artery surgery

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    Kalender G

    2016-12-01

    Full Text Available G Kalender,1 A Kornberger,2 M Lisy,1 Andres Beiras-Fernandez,2 UA Stock2 1Deparment of General, Thoracic and Vascular Surgery, Hoechst Hospital, 2Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt am Main, Germany Aim: Endothelial progenitor cells (EPCs are primitive cells found in the bone marrow and peripheral blood (PB. In particular, the potential of EPCs to differentiate into mature endothelial cells remains of high interest for clinical applications such as bio-functionalized patches for autologous seeding after implantation. The objective of this study was to determine EPCs’ kinetics in patients undergoing carotid artery thromboendarterectomy (CTEA and patch angioplasty. Methods: Twenty CTEA patients were included (15 male, mean age 76 years. PB samples were taken at 1 day preoperatively, and at 1, 3, and 5 days postoperatively. Flow cytometric analysis was performed for CD34, CD133, KDR, and CD45. Expression of KDR, SDF-1α, and G-CSF was analyzed by means of enzyme-linked immunosorbent assay. Results: Fluorescence-activated cell sorting analysis revealed 0.031%±0.016% (% of PB mononuclear cells KDR+ cells and 0.052%±0.022% CD45-/CD34+/CD133+ cells, preoperatively. A 33% decrease of CD45–/CD34+/CD133+ cells was observed at day 1 after surgery. However, a relative number (compared to initial preoperative values of CD45-/CD34+/CD133+ cells was found on day 3 (82% and on day 5 (94% postoperatively. More profound upregulated levels of CD45–CD34+/CD133+ cells were observed for diabetic (+47% compared to nondiabetic and male (+38% compared to female patients. No significant postoperative time-dependent differences were found in numbers of KDR+ cells and the concentrations of the cytokines KDR and G-CSF. However, the SDF-1α levels decreased significantly on day 1 postoperatively but returned to preoperative levels by day 3. Conclusion: CTEA results in short-term downregulation of circulating

  19. Prognostic Impact of Circulating Tumor Cell Detected Using a Novel Fluidic Cell Microarray Chip System in Patients with Breast Cancer

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    Takeshi Sawada

    2016-09-01

    Full Text Available Various types of circulating tumor cell (CTC detection systems have recently been developed that show a high CTC detection rate. However, it is a big challenge to find a system that can provide better prognostic value than CellSearch in head-to-head comparison. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC that captures CTC independently of tumor-specific markers or physical properties. Here, we compared the CTC detection sensitivity and the prognostic value of FCMC with CellSearch in breast cancer patients. FCMC was validated in preclinical studies using spike-in samples and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p = 0.010 of MDA-MB-231 cells and an equivalent detection rate (p = 0.497 of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p = 0.00037. CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by progression free survival. The hazard ratio between CTC+ and CTC− was 4.229 in CellSearch (95% CI, 1.31 to 13.66; p = 0.01591; in contrast, it was 11.31 in FCMC (95% CI, 2.245 to 57.0; p = 0.000244. CTC detected using FCMC, like the CTC detected using CellSearch, have the potential to be a strong prognostic factor for cancer patients.

  20. CCR 20th Anniversary Commentary: Circulating Tumor Cells in Prostate Cancer.

    Science.gov (United States)

    Mehra, Niven; Zafeiriou, Zafeiris; Lorente, David; Terstappen, Leon W M M; de Bono, Johann S

    2015-11-15

    Circulating tumor cells (CTC) have substantial promise for multipurpose biomarker studies in prostate cancer. The IMMC-38 trial conducted by de Bono and colleagues, which was published in the October 1, 2008, issue of Clinical Cancer Research, demonstrated for the first time that CTCs are the most accurate and independent predictor of overall survival in metastatic prostate cancer. Since the publication of prospective trials demonstrating prognostic utility, CTCs have been utilized for nucleic acid analyses, for protein analyses, and in intermediate endpoint studies. CTC studies are also now facilitating the analysis of intrapatient heterogeneity. See related article by de Bono et al., Clin Cancer Res 2008;14(19) October 1, 2008;6302-9. ©2015 American Association for Cancer Research.

  1. Considerations in the development of circulating tumor cell technology for clinical use

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    Parkinson David R

    2012-07-01

    Full Text Available Abstract This manuscript summarizes current thinking on the value and promise of evolving circulating tumor cell (CTC technologies for cancer patient diagnosis, prognosis, and response to therapy, as well as accelerating oncologic drug development. Moving forward requires the application of the classic steps in biomarker development–analytical and clinical validation and clinical qualification for specific contexts of use. To that end, this review describes methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA and the US National Cancer Institute (NCI.

  2. Feasibility of cell-free circulating tumor DNA testing for lung cancer.

    Science.gov (United States)

    Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

    2016-01-01

    Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications.

  3. Stepwise optimization of the procedure for assessment of circulating progenitor cells in patients with myocardial infarction.

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    Yu-Xin Cui

    Full Text Available The number and functional activity of circulating progenitor cells (CPCs is altered in diabetic patients. Furthermore, reduced CPC count has been shown to independently predict cardiovascular events. Validation of CPCs as a biomarker for cardiovascular risk stratification requires rigorous methodology. Before a standard operation protocol (SOP can be designed for such a trial, a variety of technical issues have to be addressed fundamentally, which include the appropriate type of red blood cell lysis buffer, FMO or isotype controls to identify rare cell populations from background noise, optimal antibody dilutions and conditions of sample storage. We herein propose improvements in critical steps of CPC isolation, antigenic characterization and determination of functional competence for final application in a prospective investigation of CPCs as a biomarker of outcome following acute myocardial infarction.In this validation study, we refined the standard operating procedure (SOP for flow cytometry characterisation and functional analysis of CPCs from the first 18 patients of the Progenitor Cell Response after Myocardial Infarction Study (ProMIS. ProMIS aims to verify the prognostic value of CPCs in patients with either ST elevation or non-ST elevation myocardial infarction with or without diabetes mellitus, using cardiac magnetic resonance imaging (MRI for assessment of ventricular function as a primary endpoint. Results indicate crucial steps for SOP implementation, namely timely cell isolation after sampling, use of appropriate lysis buffer to separate blood cell types and minimize the acquisition events during flow cytometry, adoption of proper fluorophore combination and antibody titration for multiple antigenic detection and introduction of counting beads for precise quantification of functional CPC activity in migration assay.With systematic specification of factors influencing the enumeration of CPC by flow cytometry, the abundance and

  4. Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

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    Ilaria Roato

    depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients.

  5. A novel approach for the detection and genetic analysis of live melanoma circulating tumor cells.

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    Melody J Xu

    Full Text Available Circulating tumor cell (CTC detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4% and specificity of 99.9% (95%CI 99.8-99.9%. In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

  6. Antibody-Conjugated Nontoxic Arginine-Doped Fe3O4 Nanoparticles for Magnetic Circulating Tumor Cells Separation

    OpenAIRE

    F. Kashanian; M. M. Masoudi; A. Akbari; A. Shamloo; M. R. Zand; S. S. Salehi

    2017-01-01

    Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this...

  7. Ultra-endurance exercise induces stress and inflammation and affects circulating hematopoietic progenitor cell function.

    Science.gov (United States)

    Stelzer, I; Kröpfl, J M; Fuchs, R; Pekovits, K; Mangge, H; Raggam, R B; Gruber, H-J; Prüller, F; Hofmann, P; Truschnig-Wilders, M; Obermayer-Pietsch, B; Haushofer, A C; Kessler, H H; Mächler, P

    2015-10-01

    Although amateur sports have become increasingly competitive within recent decades, there are as yet few studies on the possible health risks for athletes. This study aims to determine the impact of ultra-endurance exercise-induced stress on the number and function of circulating hematopoietic progenitor cells (CPCs) and hematological, inflammatory, clinical, metabolic, and stress parameters in moderately trained amateur athletes. Following ultra-endurance exercise, there were significant increases in leukocytes, platelets, interleukin-6, fibrinogen, tissue enzymes, blood lactate, serum cortisol, and matrix metalloproteinase-9. Ultra-endurance exercise did not influence the number of CPCs but resulted in a highly significant decline of CPC functionality after the competition. Furthermore, Epstein-Barr virus was seen to be reactivated in one of seven athletes. The link between exercise-induced stress and decline of CPC functionality is supported by a negative correlation between cortisol and CPC function. We conclude that ultra-endurance exercise induces metabolic stress and an inflammatory response that affects not only mature hematopoietic cells but also the function of the immature hematopoietic stem and progenitor cell fraction, which make up the immune system and provide for regeneration. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Circulating cell-free DNA levels increase variably following chorionic villus sampling.

    Science.gov (United States)

    Vora, Neeta L; Johnson, Kirby L; Peter, Inga; Tighiouart, Hocine; Ralston, Steven J; Craigo, Sabrina D; Bianchi, Diana W

    2010-04-01

    Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA. Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender. Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels. While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women. Copyright (c) 2010 John Wiley & Sons, Ltd.

  9. Statins Increase the Frequency of Circulating CD4+FOXP3+ Regulatory T Cells in Healthy Individuals

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    Ana Lucía Rodríguez-Perea

    2015-01-01

    Full Text Available Statins have been shown to modulate the number and the suppressive function of CD4+FOXP3+ T cells (Treg in inflammatory conditions. However, it is not well established whether statin could also affect Treg in absence of inflammation. To address this question, eighteen normocholesterolemic male subjects were treated with lovastatin or atorvastatin daily for 45 days. The frequency and phenotype of circulating Treg were evaluated at days 0, 7, 30, and 45. mRNA levels of FOXP3, IDO, TGF-β, and IL-10 were measured in CD4+ T cells. We found that both statins significantly increased Treg frequency and FOXP3 mRNA levels at day 30. At day 45, Treg numbers returned to baseline values; however, TGF-β and FOXP3 mRNA levels remained high, accompanied by increased percentages of CTLA-4- and GITR-expressing Treg. Treg Ki-67 expression was decreased upon statin treatment. Treg frequency positively correlated with plasma levels of high-density lipoprotein cholesterol (HDL-c, suggesting a role for HDL-c in Treg homeostasis. Therefore, statins appear to have inflammation-independent immune-modulatory effects. Thus, the increase in Treg cells frequency likely contributes to immunomodulatory effect of statins, even in healthy individuals.

  10. Circulation-Independent Differentiation Pathway from Extraembryonic Mesoderm toward Hematopoietic Stem Cells via Hemogenic Angioblasts

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    Yosuke Tanaka

    2014-07-01

    Full Text Available A large gap exists in our understanding of the course of differentiation from mesoderm to definitive hematopoietic stem cells (HSCs. Previously, we reported that Runx1+ cells in embryonic day 7.5 (E7.5 embryos contribute to the hemogenic endothelium in the E10.5 aorta-gonad-mesonephros (AGM region and HSCs in the adult bone marrow. Here, we show that two Runx1+ populations subdivided by Gata1 expression exist in E7.5 embryos. The hemogenic endothelium and the HSCs are derived only from the Runx1+Gata1− population. A subset of this population moves from the extra- to intraembryonic region during E7.5–E8.0, where it contributes to the hemogenic endothelium of the dorsal aorta (DA. Migration occurs before the heartbeat is initiated, and it is independent of circulation. This suggests a developmental trajectory from Runx1+ cells in the E7.5 extraembryonic region to definitive HSCs via the hemogenic endothelium.

  11. Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease.

    Science.gov (United States)

    Li, Si-Ming; Bai, Fu-Liang; Xu, Wen-Juan; Yang, Yong-Bi; An, Ying; Li, Tian-He; Yu, Yin-Hang; Li, De-Shan; Wang, Wen-Fei

    2014-09-01

    The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  12. Automated multimodal spectral histopathology for quantitative diagnosis of residual tumour during basal cell carcinoma surgery

    NARCIS (Netherlands)

    Boitor, R. (Radu); Kong, K. (Kenny); Shipp, D. (Dustin); Varma, S. (Sandeep); Koloydenko, A. (Alexey); Kulkarni, K. (Kusum); Elsheikh, S. (Somaia); Bakker Schut, T. (Tom); P.J. Caspers (Peter); G.J. Puppels (Gerwin); M.J.G. Van Der Wolf (Martin J. G.); Sokolova, E. (Elena); T.E.C. Nijsten (Tamar); Salence, B. (Brogan); H. Williams; Notingher, I. (Ioan)

    2017-01-01

    textabstractMultimodal spectral histopathology (MSH), an optical technique combining tissue auto-fluorescence (AF) imaging and Raman micro-spectroscopy (RMS), was previously proposed for detection of residual basal cell carcinoma (BCC) at the surface of surgically-resected skin tissue. Here we

  13. Effects of Flowing RBCs on Adhesion of a Circulating Tumor Cell in Microvessels

    Science.gov (United States)

    Xiao, L.L.; Liu, Y.; Chen, S.; Fu, B.M.

    2016-01-01

    Adhesion of circulating tumor cells (CTCs) to the microvessel wall largely depends on the blood hydrodynamic conditions, one of which is the blood viscosity. Since blood is a non-Newtonian fluid, whose viscosity increases with hematocrit, in the microvessels at low shear rate. In this study, the effects of hematocrit, vessel size, flow rate and red blood cells (RBCs) aggregation on adhesion of a CTC in the microvessels were numerically investigated using dissipative particle dynamics. The membrane of cells was represented by a spring-based network connected by elastic springs to characterize its deformation. RBCs aggregation was modelled by a Morse potential function based on depletion-mediated assumption and the adhesion of the CTC to the vessel wall was achieved by the interactions between receptors and ligands at the CTC and those at the endothelial cells forming the vessel wall. The results demonstrated that in the microvessel of 15μm diameter, the CTC has an increasing probability of adhesion with the hematocrit due to a growing wall-directed force, resulting in a larger number of receptor-ligand bonds formed on the cell surface. However, with the increase in microvessel size, an enhanced lift force at higher hematocrit detaches the initial adherent CTC quickly. If the microvessel is comparable to the CTC in diameter, CTC adhesion is independent of Hct. In addition, the velocity of CTC is larger than the average blood flow velocity in smaller microvessels and the relative velocity of CTC decreases with the increase in microvessel size. An increased blood flow resistance in the presence of CTC was also found. Moreover, it was found that the large deformation induced by high flow rate and the presence of aggregation promote the adhesion of CTC. PMID:27738841

  14. Tapered-slit membrane filters for high-throughput viable circulating tumor cell isolation.

    Science.gov (United States)

    Kang, Yoon-Tae; Doh, Il; Cho, Young-Ho

    2015-04-01

    This paper presents tapered-slit membrane filters for high-throughput viable circulating tumor cell (CTC) isolation. The membrane filter with a 2D array of vertical tapered slits with a gap that is wide at the entrance and gradually decreases with depth, provide minimal cell stress and reduce 82.14% of the stress generated in conventional straight-hole filters. We designed two types of tapered-slit filters, Filters 6 and 8, respectively, containing the tapered slits with outlet widths of 6 μm and 8 μm at a slit density of 34,445/cm(2) on the membrane. We fabricated the vertical slits with a tapered angle of 2 ° on a SU8 membrane by adjusting the UV expose dose and the air gap between the membrane and the photomask during lithography. In the experimental study, the proposed tapered-slit filter captured 89.87% and 82.44% of the cancer cells spiked in phosphate buffered saline (PBS) and diluted blood (blood: PBS = 1:4), respectively, at a sample flow rate of 5 ml per hour, which is 33.3 times faster than previous lateral tapered-slit filters. We further verified the capability to culture on chip after capturing: 72.33% of cells among the captured cells still remained viable after a 5-day culture. The proposed tapered-slit membrane filters verified high-throughput viable CTC isolation capability, thereby inaugurating further advanced CTC research for cancer diagnosis and prognosis.

  15. Vitamin D Status in Rheumatoid Arthritis: Inflammation, Arterial Stiffness and Circulating Progenitor Cell Number.

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    Alberto Lo Gullo

    Full Text Available Suboptimal vitamin D status was recently acknowledged as an independent predictor of cardiovascular diseases and all-cause mortality in several clinical settings, and its serum levels are commonly reduced in Rheumatoid Arthritis (RA. Patients affected by RA present accelerated atherosclerosis and increased cardiovascular morbidity and mortality with respect to the general population. In RA, it has been reported an impairment of the number and the activity of circulating proangiogenic haematopoietic cells (PHCs, including CD34+, that may play a role in endothelial homeostasis. The purpose of the study is to investigate the association between vitamin D levels and PHCs, inflammatory markers, and arterial stiffening in patients with RA.CD34+ cells were isolated from 27 RA patients and 41 controls. Vitamin D levels, C-reactive protein (CRP, fibrinogen, pulse wave velocity (PWV, and carotid intima-media thickness (cIMT were also evaluated. CD34+ count and vitamin D levels were lower in RA patients as compared to controls, while fibrinogen, CRP, PWV and cIMT were higher in RA patients. CD34+ cell number appeared to be associated with vitamin D levels, and negatively correlated to fibrinogen and early atherosclerosis markers (PWV and cIMT; vitamin D levels appear also to be inversely associated to fibrinogen.RA patients with moderate disease activity presented with low vitamin D levels, low CD34+ cell count, increased PWV and cIMT; we found that vitamin D deficiency is associated to CD34+ cell reduction in peripheral blood, and with fibrinogen levels. This suggests that vitamin D might contribute to endothelial homeostasis in patients with RA.

  16. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Galanzha, Ekaterina I. [Phillips Classic Laser and Nanomedicine Laboratories, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Zharov, Vladimir P., E-mail: zharovvladimirp@uams.edu [Phillips Classic Laser and Nanomedicine Laboratories, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Arkansas Nanomedicine Center, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2013-12-10

    Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1–10 CTC/mL) due to the small volume of blood samples (5–10 mL). Consequently, they can miss up to 10{sup 3}–10{sup 4} CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 10{sup 2}–10{sup 3} times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-resolution PAFC beyond the diffraction and spectral limits.

  17. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    Directory of Open Access Journals (Sweden)

    Ekaterina I. Galanzha

    2013-12-01

    Full Text Available Despite progress in detecting circulating tumor cells (CTCs, existing assays still have low sensitivity (1–10 CTC/mL due to the small volume of blood samples (5–10 mL. Consequently, they can miss up to 103–104 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 102–103 times by the examination of the entire blood volume in vivo (5 L in adults. We focus on in vivo photoacoustic (PA flow cytometry (PAFC of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL and throughput (up to 10 mL/min than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-rsesolution PAFC beyond the diffraction and spectral limits.

  18. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    International Nuclear Information System (INIS)

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2013-01-01

    Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1–10 CTC/mL) due to the small volume of blood samples (5–10 mL). Consequently, they can miss up to 10 3 –10 4 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 10 2 –10 3 times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-resolution PAFC beyond the diffraction and spectral limits

  19. A new one-platform flow cytometric method for residual cell counting in platelet concentrates.

    Science.gov (United States)

    Schmidt, Michael; Spengler, Hans-Peter; Lambrecht, Bernd; Hourfar, Michael K; Seifried, Erhard; Tonn, Torsten

    2009-12-01

    According to German regulations and guidelines, residual red blood cells (rRBCs) and residual white blood cells (rWBCs) must number fewer than 3 x 10(9) cells/unit and 1 x 10(6) cells/unit in platelet concentrates (PCs), respectively. Due to low levels of residual cells in final products, there is still a need for fast, reliable, and sensitive methods of automated detection of these cell types. In Part A, 21 PCs were spiked with predetermined numbers of red blood cells (RBCs) and white blood cells (WBCs). The linearity, precision, and accuracy of the BD Thrombo Count assay (BD Biosciences Europe) were tested and validated according to international guidelines. Finally in Part B, 100 PCs prepared from pooled buffy coats were tested by the BD Thrombo Count assay and compared with other methods, including Nageotte (rWBCs) and Neubauer (rRBCs) counting chambers and the flow cytometric BD LeucoCOUNT (Becton Dickinson) assay (rWBCs). The unspecific background of blank PC samples was fewer than 0.02 cells/microL for WBCs and fewer than 34 cells/microL for RBCs (mean, 21). Linear regression and precision analyses of spiked PC samples were determined for both WBCs (r(2) = 0.992; range, 0.6-6.0 WBCs/microL) and RBCs (r(2) = 0.999; 800-8000 RBCs/microL). No carryover of cells or drift in results was detected in the automated sample acquisition mode. Analysis according to statistical methods of Bland and Altman demonstrated a high correlation between BD Thrombo Count and the Neubauer manual counting chamber. This novel flow cytometric test is a quick and reliable single-tube assay that has been demonstrated as a potential alternative for the existing manual microscopic counting procedures that are both time-consuming and laborious.

  20. Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics

    International Nuclear Information System (INIS)

    Chen, Weiqiang; Allen, Steven G.; Reka, Ajaya Kumar; Qian, Weiyi; Han, Shuo; Zhao, Jianing; Bao, Liwei; Keshamouni, Venkateshwar G.; Merajver, Sofia D.; Fu, Jianping

    2016-01-01

    Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population. We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs. The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines’ metastatic ability. The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further

  1. Circulating Endothelial Cells in Patients with Heart Failure and Left Ventricular Dysfunction

    Science.gov (United States)

    Martínez-Sales, Vicenta; Sánchez-Lázaro, Ignacio; Vila, Virtudes; Almenar, Luis; Contreras, Teresa; Reganon, Edelmiro

    2011-01-01

    Introduction and Aims: Acute and chronic heart failure may manifest different degrees of endothelial damage and angiogenesis. Circulating endothelial cells (CEC) have been identified as marker of vascular damage. The aim of our study was to evaluate the evolution of the CEC at different stages of patients with heart failure. We also investigated a potential correlation between CEC and markers of vascular damage and angiogenesis. Methods: We studied 32 heart failure patients at hospital admission (acute phase) and at revision after 3 months (stable phase) and 32 controls. Circulating markers of endothelial damage (CEC; von Willebrand factor, vWF and soluble E-selectin, sEsel) and angiogenesis (vascular endothelial growth factor, VEGF and thrombospondin-1) were quantified. Results: Levels of CEC, vWF, sEsel and VEGF are significantly higher in heart failure patients than in controls. Levels of CEC (36.9 ± 15.3 vs. 21.5 ± 10.0 cells/ml; p < 0.001), vWF (325 ± 101 vs. 231 ± 82%; p < 0.001) and VEGF (26.3 ± 15.2 vs. 21.9 ± 11.9 ng/ml; p < 0.001) are significantly higher in the acute phase than in the stable phase of heart failure. CEC levels correlate with vWF and VEGF. Results show than 100% of patients in acute phase and 37.5% in stable phase have levels of CEC higher than the 99th percentile of the distribution of controls (16 cells/ml). Therefore, increases in CEC represent a relative risk of 9.5 for heart failure patients suffering from acute phase. Conclusions: CEC, in addition to being elevated in heart failure, correlate with vWF levels, providing further support for CEC as markers of endothelial damage. Levels of CEC are associated with the acute phase of heart failure and could be used as a marker of the worsening in heart failure. PMID:21897001

  2. A liquid biopsy-based method for the detection and quantification of circulating tumor cells in surgical osteosarcoma patients

    NARCIS (Netherlands)

    Zhang, Haoqiang; Gao, Peng; Xiao, Xin; Heger, Michal; Geng, Lei; Fan, Bo; Yuan, Yulin; Huang, Chen; Chen, Guojing; Liu, Yao; Hu, Yongchen; Yu, Xiuchun; Wu, Sujia; Wang, Ling; Wang, Zhen

    2017-01-01

    A method for the enumeration and quantification of osteosarcoma (OS) circulating tumor cells (CTCs) is currently not available. A correlation between the number of CTCs and progression-free survival (PFS) has been established for other cancers, but not for OS CTCs. A method was therefore developed

  3. Circulating intercellular adhesion molecule-1 (ICAM-1) as an early and sensitive marker for virus-induced T cell activation

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Johansen, J; Marker, O

    1995-01-01

    The effect of systemic virus infection on the level of circulating ICAM-1 (cICAM-1) in serum, and the role of virus-activated T cells in this context, were studied using the murine lymphocytic choriomeningitis virus infection as primary model system. A marked virus-induced elevation in cICAM-1...

  4. Circulating immune cell subpopulations in pestivirus persistently infected calves and non-infected calves varying in immune status.

    Science.gov (United States)

    Circulating immune cell subpopulations in cattle representing varying stages of immune status categorized as; colostrum deprived (CD), receiving colostrum (COL), colostrum plus vaccination (VAC) and persistently infected with a pestivirus (PI) were compared. The PI calves were infected with a HoBi-...

  5. Circulating immune cell subpopulations in pestivirus persistently infected calves and non-infected calves varying in immune status [Abstract

    Science.gov (United States)

    The circulating immune cell subpopulations in cattle representing varying stages of immune status categorized as; colostrum deprived (CD), receiving colostrum (COL), colostrum plus vaccination (VAC) and persistently infected with a pestivirus (PI) were compared. The PI calves were infected with a H...

  6. EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Helen Schneck

    Full Text Available Circulating tumor cells (CTCs are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM. But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT. Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47 was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3 and EpCAMlow/neg (MDA-MB-231 breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA, collagen I, laminin for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69% from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. Ep

  7. EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer

    Science.gov (United States)

    Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H.; Templin, Markus F.; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans

    2015-01-01

    Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far–including the gold standard CellSearch—rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1–24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. Ep

  8. Relationship between circulating tumor cells and epithelial to mesenchymal transition in early breast cancer

    International Nuclear Information System (INIS)

    Mego, M.; Cierna, Z.; Janega, P.; Karaba, M.; Minarik, G.; Benca, J.; Sedlácková, T.; Sieberova, G.; Gronesova, P.; Manasova, D.; Pindak, D.; Sufliarsky, J.; Danihel, L.; Reuben, JM; Mardiak, J.

    2015-01-01

    Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. Epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue. This study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. Expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score. CTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT. In this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features

  9. DECREASED LEVEL OF CORD BLOOD CIRCULATING ENDOTHELIAL COLONY-FORMING CELLS IN PREECLAMPSIA

    Science.gov (United States)

    Muñoz-Hernandez, Rocio; Miranda, Maria L.; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M.; Dominguez-Simeon, Maria J.; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M.

    2014-01-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation and migration towards VEGF-A and FGF-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P = .04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events. PMID:24752434

  10. Integration of biomimicry and nanotechnology for significantly improved detection of circulating tumor cells (CTCs).

    Science.gov (United States)

    Myung, Ja Hye; Park, Sin-Jung; Wang, Andrew Z; Hong, Seungpyo

    2017-12-13

    Circulating tumor cells (CTCs) have received a great deal of scientific and clinical attention as a biomarker for diagnosis and prognosis of many types of cancer. Given their potential significance in clinics, a variety of detection methods, utilizing the recent advances in nanotechnology and microfluidics, have been introduced in an effort of achieving clinically significant detection of CTCs. However, effective detection and isolation of CTCs still remain a tremendous challenge due to their extreme rarity and phenotypic heterogeneity. Among many approaches that are currently under development, this review paper focuses on a unique, promising approach that takes advantages of naturally occurring processes achievable through application of nanotechnology to realize significant improvement in sensitivity and specificity of CTC capture. We provide an overview of successful outcome of this biomimetic CTC capture system in detection of tumor cells from in vitro, in vivo, and clinical pilot studies. We also emphasize the clinical impact of CTCs as biomarkers in cancer diagnosis and predictive prognosis, which provides a cost-effective, minimally invasive method that potentially replaces or supplements existing methods such as imaging technologies and solid tissue biopsy. In addition, their potential prognostic values as treatment guidelines and that ultimately help to realize personalized therapy are discussed. Copyright © 2017. Published by Elsevier B.V.

  11. Circulating Tumor Cells: What Is in It for the Patient? A Vision towards the Future

    International Nuclear Information System (INIS)

    Stolpe, Anja van de; Toonder, Jaap M. J. den

    2014-01-01

    Knowledge on cellular signal transduction pathways as drivers of cancer growth and metastasis has fuelled development of “targeted therapy” which “targets” aberrant oncogenic signal transduction pathways. These drugs require nearly invariably companion diagnostic tests to identify the tumor-driving pathway and the cause of the abnormal pathway activity in a tumor sample, both for therapy response prediction as well as for monitoring of therapy response and emerging secondary drug resistance. Obtaining sufficient tumor material for this analysis in the metastatic setting is a challenge, and circulating tumor cells (CTCs) may provide an attractive alternative to biopsy on the premise that they can be captured from blood and the companion diagnostic test results are correctly interpreted. We discuss novel companion diagnostic directions, including the challenges, to identify the tumor driving pathway in CTCs, which in combination with a digital pathology platform and algorithms to quantitatively interpret complex CTC diagnostic results may enable optimized therapy response prediction and monitoring. In contrast to CTC-based companion diagnostics, CTC enumeration is envisioned to be largely replaced by cell free tumor DNA measurements in blood for therapy response and recurrence monitoring. The recent emergence of novel in vitro human model systems in the form of cancer-on-a-chip may enable elucidation of some of the so far elusive characteristics of CTCs, and is expected to contribute to more efficient CTC capture and CTC-based diagnostics

  12. Circulating Tumor Cells: What Is in It for the Patient? A Vision towards the Future

    Directory of Open Access Journals (Sweden)

    Anja van de Stolpe

    2014-05-01

    Full Text Available Knowledge on cellular signal transduction pathways as drivers of cancer growth and metastasis has fuelled development of “targeted therapy” which “targets” aberrant oncogenic signal transduction pathways. These drugs require nearly invariably companion diagnostic tests to identify the tumor-driving pathway and the cause of the abnormal pathway activity in a tumor sample, both for therapy response prediction as well as for monitoring of therapy response and emerging secondary drug resistance. Obtaining sufficient tumor material for this analysis in the metastatic setting is a challenge, and circulating tumor cells (CTCs may provide an attractive alternative to biopsy on the premise that they can be captured from blood and the companion diagnostic test results are correctly interpreted. We discuss novel companion diagnostic directions, including the challenges, to identify the tumor driving pathway in CTCs, which in combination with a digital pathology platform and algorithms to quantitatively interpret complex CTC diagnostic results may enable optimized therapy response prediction and monitoring. In contrast to CTC-based companion diagnostics, CTC enumeration is envisioned to be largely replaced by cell free tumor DNA measurements in blood for therapy response and recurrence monitoring. The recent emergence of novel in vitro human model systems in the form of cancer-on-a-chip may enable elucidation of some of the so far elusive characteristics of CTCs, and is expected to contribute to more efficient CTC capture and CTC-based diagnostics.

  13. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma.

    Science.gov (United States)

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard; Handberg, Aase

    2014-01-01

    Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer. We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men. We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

  14. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Science.gov (United States)

    Sharon, Eilon; Shi, Hao; Kharbanda, Sandhya; Koh, Winston; Martin, Lance R; Khush, Kiran K; Valantine, Hannah; Pritchard, Jonathan K; De Vlaminck, Iwijn

    2017-08-01

    Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  15. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    2017-08-01

    Full Text Available Quantification of cell-free DNA (cfDNA in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD quantifies donor-derived cfDNA (dd-cfDNA by taking advantage of single-nucleotide polymorphisms (SNPs distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM of identity-by-descent (IBD states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD. These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  16. Fourier ptychographic microscopy for filtration-based circulating tumor cell enumeration and analysis.

    Science.gov (United States)

    Williams, Anthony; Chung, Jaebum; Ou, Xiaoze; Zheng, Guoan; Rawal, Siddarth; Ao, Zheng; Datar, Ram; Yang, Changhuei; Cote, Richard

    2014-06-01

    Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM's ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R² = 0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.

  17. Quantitative detection of residual E. coli host cell DNA by real-time PCR.

    Science.gov (United States)

    Lee, Dong Hyuck; Bae, Jung Eun; Lee, Jung Hee; Shin, Jeong Sup; Kim, In Seop

    2010-10-01

    E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.

  18. Circulating gluten-specific FOXP3+CD39+regulatory T cells have impaired suppressive function in patients with celiac disease.

    Science.gov (United States)

    Cook, Laura; Munier, C Mee Ling; Seddiki, Nabila; van Bockel, David; Ontiveros, Noé; Hardy, Melinda Y; Gillies, Jana K; Levings, Megan K; Reid, Hugh H; Petersen, Jan; Rossjohn, Jamie; Anderson, Robert P; Zaunders, John J; Tye-Din, Jason A; Kelleher, Anthony D

    2017-12-01

    Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3) + Treg cells. Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4 + T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4 + T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4 + T cells were FOXP3 + CD39 + Treg cells, which reside within the pool of memory CD4 + CD25 + CD127 low CD45RO + Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3 + CD39 + Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. This study provides the first estimation of FOXP3 + CD39 + Treg cell frequency within circulating gluten-specific CD4 + T cells after oral gluten challenge of patients with celiac disease. FOXP3 + CD39 + Treg cells comprised a major proportion of all circulating gluten-specific CD4 + T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key

  19. Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

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    Jeehye Maeng

    2015-04-01

    Full Text Available Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

  20. The construction of a Pomeranchuk cell driven by a 4He-circulating dilution refrigerator and some related experiments

    International Nuclear Information System (INIS)

    Brandt, B. van den.

    1981-01-01

    This thesis deals with some investigations at very low temperatures in which a 4 He-circulating 3 He- 4 He dilution refrigerator and a Pomeranchuk cooling device are used. The main theme is the design and construction of a special device, a Pomeranchuk cell that is precooled and pressurized with a 4 He-circulating 3 He- 4 He dilution refrigerator, that can be used to obtain a lowest temperature of the order of 1.1 mK. Furthermore, some details of the working of such a dilution refrigerator and some properties of 3 He- 4 He mixtures at high pressures were investigated. (Auth.)

  1. Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

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    Mirick Gary

    2009-04-01

    Full Text Available Abstract Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL (DvLPBaPPP2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a

  2. Circulating cell death products predict clinical outcome of colorectal cancer patients

    International Nuclear Information System (INIS)

    Koelink, Pim J; Lamers, Cornelis BHW; Hommes, Daan W; Verspaget, Hein W

    2009-01-01

    Tumor cell death generates products that can be measured in the circulation of cancer patients. CK18-Asp396 (M30 antigen) is a caspase-degraded product of cytokeratin 18 (CK18), produced by apoptotic epithelial cells, and is elevated in breast and lung cancer patients. We determined the CK18-Asp396 and total CK18 levels in plasma of 49 colorectal cancer patients, before and after surgical resection of the tumor, by ELISA. Correlations with patient and tumor characteristics were determined by Kruskal-Wallis H and Mann-Whitney U tests. Disease-free survival was determined using Kaplan-Meier methodology with Log Rank tests, and univariate and multivariate Cox proportional hazard analysis. Plasma CK18-Asp396 and total CK18 levels in colorectal cancer patients were related to disease stage and tumor diameter, and were predictive of disease-free survival, independent of disease-stage, with hazard ratios (HR) of patients with high levels (> median) compared to those with low levels (≤ median) of 3.58 (95% CI: 1.17–11.02) and 3.58 (95% CI: 0.97–7.71), respectively. The CK18-Asp396/CK18 ratio, which decreased with tumor progression, was also predictive of disease-free survival, with a low ratio (≤ median) associated with worse disease-free survival: HR 2.78 (95% CI: 1.06–7.19). Remarkably, the plasma CK18-Asp396 and total CK18 levels after surgical removal of the tumor were also predictive of disease-free survival, with patients with high levels having a HR of 3.78 (95% CI: 0.77–18.50) and 4.12 (95% CI: 0.84–20.34), respectively, indicating that these parameters can be used also to monitor patients after surgery. CK18-Asp396 and total CK18 levels in the circulation of colorectal cancer patients are predictive of tumor progression and prognosis and might be helpful for treatment selection and monitoring of these patients

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

    Science.gov (United States)

    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.

  4. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

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    Morten Hjuler Nielsen

    2014-02-01

    Full Text Available Background and aim: Previous studies on circulating microparticles (MPs indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer. Methods: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females. MPs were identified according to their size (<1.0-µm, by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS exposing MPs binding Lactadherin was determined. Results: By using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men. Conclusion: We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

  5. Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method.

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    Cecilia Bozzetti

    Full Text Available The exclusion of circulating tumor cells (CTCs that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT process by using Epithelial Cell Adhesion Molecule (EpCAM based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile.Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1 probe and by immunocytochemistry. Thirty healthy donors were also tested.Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89% SQCLC patients. Thirty-four of the 44 (77% samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42 of pooled circulating SQCLC cells, whereas only 29% (14/48 were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found.Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the

  6. Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method

    Science.gov (United States)

    Bozzetti, Cecilia; Quaini, Federico; Squadrilli, Anna; Tiseo, Marcello; Frati, Caterina; Lagrasta, Costanza; Azzoni, Cinzia; Bottarelli, Lorena; Galetti, Maricla; Alama, Angela; Belletti, Silvana; Gatti, Rita; Passaro, Antonio; Gradilone, Angela; Cavazzoni, Andrea; Alfieri, Roberta; Petronini, Pier Giorgio; Bonelli, Mara; Falco, Angela; Carubbi, Cecilia; Pedrazzi, Giuseppe; Nizzoli, Rita; Naldi, Nadia; Pinto, Carmine; Ardizzoni, Andrea

    2015-01-01

    Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved

  7. Assessment of the immunogenicity of residual host cell protein impurities of OsrHSA.

    Science.gov (United States)

    Abiri, Naghmeh; Pang, Jianlei; Ou, Jiquan; Shi, Bo; Wang, Xianghong; Zhang, Sucai; Sun, Yunxia; Yang, Daichang

    2018-01-01

    Human serum albumin (HSA) is the most abundant protein in human plasma and is widely used at high doses for treating various diseases. Recombinant HSA is an alternative approach to plasma-derived HSA, providing increased safety and an unlimited supply. However, the safety of the residual host cell proteins (HCPs) co-purified with Oryza sativa HSA (OsrHSA) remains to be determined. An animal system was used to assess the immunogenicity of OsrHSA and its residual HCPs. Low immunogenicity and immunotoxicity of the residual HCPs at a dose of 25 μg/kg, equivalent to 25 times the clinical dosage of HSA, were observed. An anti-drug-antibody (ADA) analysis revealed that anti-HSA, anti-OsrHSA or anti-HCP antibodies developed with a low frequency in pHSA and OsrHSA treatments, but the titers were as low as 1.0-2.0. Furthermore, the titer and the incidence of the specific antibodies were not significantly different between the pHSA and OsrHSA groups, indicating that OsrHSA presents similar immunogenicity to that of pHSA. More importantly, no cytokines were stimulated after the administration of OsrHSA and the residual HCPs, suggesting that there was no risk of a cytokine storm. These results demonstrated that the residual HCPs from OsrHSA have low immunogenicity, indicating that the rice endosperm is one of the best hosts for plant molecular pharming.

  8. Clinical significance of the molecular detection of melanoma cells circulating in the peripheral blood in melanoma patients.

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    Konstantopoulos, K; Psatha, M; Kalotychou, V; Frangia, N; Ioannovits, I; Meletis, I; Loukopoulos, D

    2001-06-01

    Blood circulating melanoma cells may be important for the spread of the disease. The current methods are not sensitive in detecting micro metastases. Tyrosinase mRNA can be detected in peripheral blood by a molecular test. As tyrosinase is expressed only in melanocytes and melanocytes normally do not circulate in the blood, the test may prove reliable in detecting circulating melanoma cells. we used a reverse-transcription polymerase chain reaction (RT-PCR) detecting tyrosinase mRNA in the blood. A prospective investigation in melanoma patients undergoing surgery was conducted; follow-up duration was 12 months. University Department Laboratory and Melanoma Clinic of a Tertiary Hospital. a total of 27 Greek patients with a diagnosis of malignant melanoma at different stages of the disease; 12 months follow-up after surgery. Samples form 12 healthy volunteers and 13 patients with chronic myelogenous leukemia served as controls. none. none. We detected mRNA tyrosinase in the peripheral blood in 16 out of 27 melanoma patients studied. No tyrosinase mRNA was detected in any of the 25 samples from the controls. Two of the 16 positive cases developed a metastasis within the next 12 months following testing. The other 14 positive cases remain metastasis free for this period, as also did the test negative cases. Detection of blood circulating melanoma cells by a RT-PCR technique, may be helpful in defining melanoma patients who are at risk for the spread of the disease.

  9. Expression of E-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways?

    International Nuclear Information System (INIS)

    Burdick, Monica M.; Henson, Karissa A.; Delgadillo, Luis F.; Choi, Young Eun; Goetz, Douglas J.; Tees, David F. J.; Benencia, Fabian

    2012-01-01

    Although significant progress has been made in the fight against cancer, successful treatment strategies have yet to be developed to combat those tumors that have metastasized to distant organs. Poor characterization of the molecular mechanisms of cancer spread is a major impediment to designing predictive diagnostics and effective clinical interventions against late stage disease. In hematogenous metastasis, it is widely suspected that circulating tumor cells (CTCs) express specific adhesion molecules that actively initiate contact with the vascular endothelium lining the vessel walls of the target organ. This “tethering” is mediated by ligands expressed by CTCs that bind to E-selectin expressed by endothelial cells. However, it is currently unknown whether expression of functional E-selectin ligands on CTCs is related to cancer stem cell regulatory or maintenance pathways, particularly epithelial-to-mesenchymal transition and the reverse, mesenchymal-to-epithelial transition. In this hypothesis and theory article, we explore the potential roles of these mechanisms on the dynamic regulation of selectin ligands mediating CTC trafficking during metastasis.

  10. A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

    Science.gov (United States)

    Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

    2017-09-01

    The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

  11. Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues.

    Science.gov (United States)

    Akşit, Dilek; Yazıcı, Alper; Akşit, Hasan; Sarı, Esin S; Yay, Arzu; Yıldız, Onur; Kılıç, Adil; Ermiş, Sıtkı S; Seyrek, Kamil

    2016-07-01

    Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. Animal experiment. Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA) strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues) specific Griffonia simplicifolia-1 (GSL-1) was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc) specific wheat germ agglutinin (WGA) was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells.

  12. Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues

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    Dilek Akşit

    2016-08-01

    Full Text Available Background: Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. Aims: The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. Study Design: Animal experiment. Methods: Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. Results: Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues specific Griffonia simplicifolia-1 (GSL–1 was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc specific wheat germ agglutinin (WGA was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. Conclusion: Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells.

  13. Prognostic value of circulating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma

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    Qiao Y

    2017-03-01

    Full Text Available Yuanyuan Qiao,1,* Jun Li,2,* Chenghe Shi,1,* Wei Wang,2 Xiuhua Qu,1 Ming Xiong,1 Yulin Sun,3 Dandan Li,1 Xiaohang Zhao,1,3 Dajin Zhang1 1Center of Basic Medical Sciences, 2Department of Thoracic Surgery, Navy General Hospital of Chinese PLA, 3State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China *These authors contributed equally to this work Objective: Circulating tumor cells (CTCs of patients with malignant tumors can be used as a prognostic marker. However, there are few relevant reports to date on esophageal squamous cell carcinoma (ESCC. Our study assesses the clinical significance of CTCs in ESCC patients. Patients and methods: CTCs were detected in 103 peripheral blood (PB samples from 59 ESCC patients. Correlation between CTCs and clinical parameters was analyzed using the χ2 test or Fisher’s exact test. Overall survival (OS and progression-free survival (PFS were analyzed using Kaplan–Meier analysis and univariate and multivariate methods. Results: The CTC detection rate was 79.7% (47/59 at baseline. The frequency of CTC-positive patients increased as the disease stage advanced (88.0% in stages III–IV, 58.9% in stages I–II. CTC counts ≥0/7.5 mL of PB were correlated with the degree of tumor differentiation, tumor infiltration, and lymph node and distant metastases. Overall, the OS and PFS of patients with CTC counts ≥3 or ≥5/7.5 mL of PB before surgery were significantly shorter than those of patients with CTC counts <3 or <5/7.5 mL. Multivariate analysis showed CTC counts ≥5/7.5 mL of PB to be a strong prognostic indicator of OS (hazard ratio [HR] 12.478; 95% confidence interval [CI], 8.2–34.3; P<0.05 and PFS (HR 6.524; 95% CI, 1.2–34.3; P<0.05 in ESCC patients. Patients in whom CTCs changed from positive at baseline to a negative value after surgery had an excellent prognosis

  14. Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

    Science.gov (United States)

    Vietsch, Eveline E; Graham, Garrett T; McCutcheon, Justine N; Javaid, Aamir; Giaccone, Giuseppe; Marshall, John L; Wellstein, Anton

    2017-12-01

    Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Chronic stress is associated with reduced circulating hematopoietic progenitor cell number: A maternal caregiving model.

    Science.gov (United States)

    Aschbacher, Kirstin; Milush, Jeffrey M; Gilbert, Amanda; Almeida, Carlos; Sinclair, Elizabeth; Epling, Lorrie; Grenon, S Marlene; Marco, Elysa J; Puterman, Eli; Epel, Elissa

    2017-01-01

    Chronic psychological stress is a risk factor for cardiovascular disease and mortality. Circulating hematopoietic progenitor cells (CPCs) maintain vascular homeostasis, correlate with preclinical atherosclerosis, and prospectively predict cardiovascular events. We hypothesize that (1) chronic caregiving stress is related to reduced CPC number, and (2) this may be explained in part by negative interactions within the family. We investigated levels of stress and CPCs in 68 healthy mothers - 31 of these had children with an autism spectrum disorder (M-ASD) and 37 had neurotypical children (M-NT). Participants provided fasting blood samples, and CD45 + CD34 + KDR + and CD45 + CD133 + KDR + CPCs were assayed by flow cytometry. We averaged the blom-transformed scores of both CPCs to create one index. Participants completed the perceived stress scale (PSS), the inventory for depressive symptoms (IDS), and reported on daily interactions with their children and partners, averaged over 7 nights. M-ASD exhibited lower CPCs than M-NT (Cohen's d=0.83; p⩽0.01), controlling for age, BMI, and physical activity. Across the whole sample, positive interactions were related to higher CPCs, and negative interactions to lower CPCs (allp'scaregivers, child-related interpersonal stress appears to be a key psychological predictor of stress-related CVD risk. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Can Biomarker Assessment on Circulating Tumor Cells Help Direct Therapy in Metastatic Breast Cancer?

    Energy Technology Data Exchange (ETDEWEB)

    Turner, Natalie [Sandro Pitigliani Medical Oncology Department, Prato Hospital, Istituto Toscano Tumori, Via Ugo Foscolo, Prato, PO 59100 (Italy); Pestrin, Marta [Sandro Pitigliani Medical Oncology Department, Prato Hospital, Istituto Toscano Tumori, Via Ugo Foscolo, Prato, PO 59100 (Italy); Translational Research Laboratory, Prato Hospital, Via Ugo Foscolo, Prato, PO 59100 (Italy); Galardi, Francesca; De Luca, Francesca [Translational Research Laboratory, Prato Hospital, Via Ugo Foscolo, Prato, PO 59100 (Italy); Malorni, Luca [Sandro Pitigliani Medical Oncology Department, Prato Hospital, Istituto Toscano Tumori, Via Ugo Foscolo, Prato, PO 59100 (Italy); Translational Research Laboratory, Prato Hospital, Via Ugo Foscolo, Prato, PO 59100 (Italy); Di Leo, Angelo, E-mail: adileo@usl4.toscana.it [Sandro Pitigliani Medical Oncology Department, Prato Hospital, Istituto Toscano Tumori, Via Ugo Foscolo, Prato, PO 59100 (Italy)

    2014-03-25

    Circulating tumor cell (CTC) count has prognostic significance in metastatic breast cancer, but the predictive utility of CTCs is uncertain. Molecular studies on CTCs have often been limited by a low number of CTCs isolated from a high background of leukocytes. Improved enrichment techniques are now allowing molecular characterisation of single CTCs, whereby molecular markers on single CTCs may provide a real-time assessment of tumor biomarker status from a blood test or “liquid biopsy”, potentially negating the need for a more invasive tissue biopsy. The predictive ability of CTC biomarker analysis has predominantly been assessed in relation to HER2, with variable and inconclusive results. Limited data exist for other biomarkers, such as the estrogen receptor. In addition to the need to define and validate the most accurate and reproducible method for CTC molecular analysis, the clinical relevance of biomarkers, including gain of HER2 on CTC after HER2 negative primary breast cancer, remains uncertain. This review summarises the currently available data relating to biomarker evaluation on CTCs and its role in directing management in metastatic breast cancer, discusses limitations, and outlines measures that may enable future development of this approach.

  17. Chicken primordial germ cells use the anterior vitelline veins to enter the embryonic circulation

    Directory of Open Access Journals (Sweden)

    Ana De Melo Bernardo

    2012-09-01

    During gastrulation, chicken primordial germ cells (PGCs are present in an extraembryonic region of the embryo from where they migrate towards the genital ridges. This is also observed in mammals, but in chicken the vehicle used by the migratory PGCs is the vascular system. We have analysed the migratory pathway of chicken PGCs, focusing on the period of transition from the extraembryonic region to the intraembryonic vascular system. Our findings show that at Hamburger and Hamilton developmental stage HH12–HH14 the majority of PGCs concentrate axially in the sinus terminalis and favour transport axially via the anterior vitelline veins into the embryonic circulation. Moreover, directly blocking the blood flow through the anterior vitelline veins resulted in an accumulation of PGCs in the anterior region and a decreased number of PGCs in the genital ridges. We further confirmed the key role for the anterior vitelline veins in the correct migration of PGCs using an ex ovo culture method that resulted in defective morphogenetic development of the anterior vitelline veins. We propose a novel model for the migratory pathway of chicken PGCs whereby the anterior vitelline veins play a central role at the extraembryonic and embryonic interface. The chicken model of PGC migration through the vasculature may be a powerful tool to study the process of homing (inflammation and metastasis due to the striking similarities in regulatory signaling pathways (SDF1–CXCR4 and the transient role of the vasculature.

  18. Recombinant human granulocyte colony-stimulating factor increases circulating CD34-postive cells in patients with AIDS

    DEFF Research Database (Denmark)

    Nielsen, S D; Dam-Larsen, S; Nielsen, C

    1997-01-01

    In a gene therapy-based treatment of AIDS, it would be desirable to have as many transduced target cells as possible. A limiting factor is the number of target cells. In this study, we investigated whether it was possible to increase the absolute number of one possible target cell, i.......e., the circulating hematopoietic progenitor cells (CD34 cells) in patients with AIDS, using the recombinant human granulocyte colony-stimulating factor (G-CSF). Eight patients with AIDS were treated with G-CSF for neutropenia (... cells. The median increase in CD34 cells was from 0.8 to 2.2 x 10(6)/l. Finally, using a highly sensitive HIV-1 RNA PCR, we found that treatment of AIDS patients with G-CSF did not lead to enhanced HIV replication. These observations indicate that G-CSF may be used to mobilize CD34 cells in patients...

  19. Late Release of Circulating Endothelial Cells and Endothelial Progenitor Cells after Chemotherapy Predicts Response and Survival in Cancer Patients

    Directory of Open Access Journals (Sweden)

    Jeanine M. Roodhart

    2010-01-01

    Full Text Available We and others have previously demonstrated that the acute release of progenitor cells in response to chemotherapy actually reduces the efficacy of the chemotherapy. Here, we take these data further and investigate the clinical relevance of circulating endothelial (progenitor cells (CE(PCs and modulatory cytokines in patients after chemotherapy with relation to progression-free and overall survival (PFS/OS. Patients treated with various chemotherapeutics were included. Blood sampling was performed at baseline, 4 hours, and 7 and 21 days after chemotherapy. The mononuclear cell fraction was analyzed for CE(PC by FACS analysis. Plasma was analyzed for cytokines by ELISA or Luminex technique. CE(PCs were correlated with response and PFS/OS using Cox proportional hazard regression analysis. We measured CE(PCs and cytokines in 71 patients. Only patients treated with paclitaxel showed an immediate increase in endothelial progenitor cell 4 hours after start of treatment. These immediate changes did not correlate with response or survival. After 7 and 21 days of chemotherapy, a large and consistent increase in CE(PC was found (P < .01, independent of the type of chemotherapy. Changes in CE(PC levels at day 7 correlated with an increase in tumor volume after three cycles of chemotherapy and predicted PFS/OS, regardless of the tumor type or chemotherapy. These findings indicate that the late release of CE(PC is a common phenomenon after chemotherapeutic treatment. The correlation with a clinical response and survival provides further support for the biologic relevance of these cells in patients' prognosis and stresses their possible use as a therapeutic target.

  20. The ratio of circulating regulatory T cells (Tregs/Th17 cells is associated with acute allograft rejection in liver transplantation.

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    Ying Wang

    Full Text Available CD4(+CD25(+FoxP3(+ regulatory T cells (Tregs and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR (n = 16 and stable allograft liver function group (Gr-SF (n = 22. The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI after liver transplantation. In addition, the percentages of CTLA-4(+, HLA-DR(+, Ki67(+, and IL-10(+ Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.

  1. Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus.

    Directory of Open Access Journals (Sweden)

    Ayan Dey

    Full Text Available The "gold standard" for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV. This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs as a potential means to evaluate mucosal immunity to poliovirus vaccine.199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV, bivalent OPV (bOPV, or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge, and on days 31, 35 and 42 and processed for poliovirus isolation.An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001. In the IPV group, α4β7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus

  2. Correlation of circulating MMP-9 with white blood cell count in humans: effect of smoking.

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    Soren Snitker

    Full Text Available Matrix metalloproteinase-9 (MMP-9 is an emerging biomarker for several disease conditions, where white blood cell (WBC count is also elevated. In this study, we examined the relationship between MMP-9 and WBC levels in apparently healthy smoking and non-smoking human subjects.We conducted a cross-sectional study to assess the relationship of serum MMP-9 with WBC in 383 men and 356 women. Next, we divided the male population (women do not smoke in this population into three groups: never (n = 243, current (n = 76 and former (n = 64 smokers and compared the group differences in MMP-9 and WBC levels and their correlations within each group.Circulating MMP-9 and WBC count are significantly correlated in men (R(2 = 0.13, p<0.001 and women (R(2 = 0.19, p<0.001. After stratification by smoking status, MMP-9 level was significantly higher in current smokers (mean ± SE; 663.3±43.4 ng/ml, compared to never (529.7±20.6 and former smokers (568±39.3. WBC count was changed in a similar pattern. Meanwhile, the relationship became stronger in current smokers with increased correlation coefficient of r = 0.45 or R(2 = 0.21 (p<0.001 and steeper slope of ß = 1.16±0.30 (p<0.001 in current smokers, compared to r = 0.26 or R(2 = 0.07 (p<0.001 and ß = 0.34±0.10 (p<0.001 in never smokers.WBC count accounts for 13% and 19% of MMP-9 variance in men and women, respectively. In non-smoking men, WBC count accounts for 7% of MMP-9 variance, but in smoking subjects, it accounts for up to 21% of MMP-9 variance. Thus, we have discovered a previously unrecognized correlation between the circulating MMP-9 and WBC levels in humans.

  3. Prognostic Significance of Circulating and Endothelial Progenitor Cell Markers in Type 2 Diabetic Foot

    Directory of Open Access Journals (Sweden)

    Maria Sambataro

    2014-01-01

    Full Text Available Objective. We studied circulating precursor cells (CPC in type 2 diabetes mellitus (T2DM with neuropathic foot lesions with or without critical limb ischemia and relationships between endothelial precursor cells (EPC and peripheral neuropathy. Methods and Subjects. We measured peripheral blood CD34, CD133, and CD45 markers for CPC and KDR, CD31 markers for EPC by citofluorimetry and systemic neural nociceptor CGRP (calcitonin gene related protein by ELISA in 8 healthy controls (C and 62 T2DM patients: 14 with neuropathy (N, 20 with neuropathic foot lesions (N1, and 28 with neuroischemic recent revascularized (N2 foot lesions. Timing of lesions was: acute (until 6 weeks, healed, and not healed. Results. CD34+ and CD133+ were reduced in N, N1, and N2 versus C, and CD34+ were lower in N2 versus N1 (P=0.03. In N2 CD34+KDR+ remain elevated in healed versus chronic lesions and, in N1 CD133+31+ were elevated in acute lesions. CGRP was reduced in N2 and N1 versus C (P<0.04 versus C 26±2 pg/mL. CD34+KDR+ correlated in N2 with oximetry and negatively in N1 with CGRP. Conclusions. CD34+ CPC are reduced in diabetes with advanced complications and diabetic foot. CD34+KDR+ and CD31+133+ EPC differentiation could have a prognostic and therapeutic significance in the healing process of neuropathic and neuroischemic lesions.

  4. Gene profiling and circulating tumor cells as biomarker to prognostic of patients with locoregional breast cancer.

    Science.gov (United States)

    Kuniyoshi, Renata K; Gehrke, Flávia de Sousa; Alves, Beatriz C A; Vilas-Bôas, Viviane; Coló, Anna E; Sousa, Naiara; Nunes, João; Fonseca, Fernando L A; Del Giglio, Auro

    2015-09-01

    The gene profile of primary tumors, as well as the identification of circulating tumor cells (CTCs), can provide important prognostic and predictive information. In this study, our objective was to perform tumor gene profiling (TGP) in combination with CTC characterization in women with nonmetastatic breast cancer. Biological samples (from peripheral blood and tumors) from 167 patients diagnosed with stage I, II, and III mammary carcinoma, who were also referred for adjuvant/neoadjuvant chemotherapy, were assessed for the following parameters: (a) the presence of CTCs identified by the expression of CK-19 and c-erbB-2 in the peripheral blood mononuclear cell (PBMC) fraction by quantitative reverse transcription PCR (RT-PCR) and (b) the TGP, which was determined by analyzing the expression of 21 genes in paraffin-embedded tissue samples by quantitative multiplex RT-PCR with the Plexor® system. We observed a statistically significant correlation between the progression-free interval (PFI) and the clinical stage (p = 0.000701), the TGP score (p = 0.006538), and the presence of hormone receptors in the tumor (p = 0.0432). We observed no correlation between the PFI and the presence or absence of CK-19 or HER2 expression in the PBMC fraction prior to the start of treatment or in the two following readouts. Multivariate analysis revealed that only the TGP score significantly correlated with the PFI (p = 0.029247). The TGP is an important prognostic variable for patients with locoregional breast cancer. The presence of CTCs adds no prognostic value to the information already provided by the TGP.

  5. Apoptotic circulating tumor cells in early and metastatic breast cancer patients.

    Science.gov (United States)

    Kallergi, Galatea; Konstantinidis, Georgios; Markomanolaki, Harris; Papadaki, Maria A; Mavroudis, Dimitris; Stournaras, Christos; Georgoulias, Vassilis; Agelaki, Sofia

    2013-09-01

    The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P = 0.0001; M30-antibody: 32.1% vs. 76.63%; P = 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics.

  6. Depletion of Treg cells inhibits minimal residual disease after surgery of HPV16-associated tumours

    Czech Academy of Sciences Publication Activity Database

    Šímová, Jana; Bubeník, Jan; Bieblová, Jana; Rosalia, Rodney Alexander; Frič, Jan; Reiniš, Milan

    2006-01-01

    Roč. 29, č. 6 (2006), s. 1567-1571 ISSN 1019-6439 R&D Projects: GA ČR(CZ) GA301/04/0492 EU Projects: European Commission(XE) 18933 - CLINIGENE Grant - others:Liga proti rakovině(CZ) XX Institutional research plan: CEZ:AV0Z50520514 Keywords : HPV16 * residual tumour disease * Treg cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.556, year: 2006

  7. HPV16-associated tumours: Therapy of surgical minimal residual disease with dendritic cell-based vaccines

    Czech Academy of Sciences Publication Activity Database

    Reiniš, Milan; Indrová, Marie; Mendoza, Luis; Mikyšková, Romana; Bieblová, Jana; Bubeník, Jan; Šímová, Jana

    2004-01-01

    Roč. 25, č. 4 (2004), s. 1165-1170 ISSN 1019-6439 R&D Projects: GA MZd NC7148; GA ČR GA301/04/0492; GA ČR GA301/01/0985; GA AV ČR IAA5052203 Institutional research plan: CEZ:AV0Z5052915 Keywords : HPV 16 * minimal residual tumour disease * dendritic cells Subject RIV: EC - Immunology Impact factor: 3.056, year: 2004

  8. Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues

    Energy Technology Data Exchange (ETDEWEB)

    Hong Jongin; DeMello, Andrew J [Nanostructured Materials and Devices Group, Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ (United Kingdom); Jayasinghe, Suwan N, E-mail: a.demello@imperial.ac.u, E-mail: s.jayasinghe@ucl.ac.u [BioPhysics Group, Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE (United Kingdom)

    2010-04-15

    Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cells) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues. (communication)

  9. Impaired circulating CD4+ LAP+ regulatory T cells in patients with acute coronary syndrome and its mechanistic study.

    Directory of Open Access Journals (Sweden)

    Zheng-Feng Zhu

    Full Text Available OBJECTIVE: CD4(+ latency-associated peptide (LAP(+ regulatory T cells (Tregs are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS has not been explored. We designed to investigate whether circulating frequency and function of CD4(+LAP(+ Tregs are defective in ACS. METHODS: One hundred eleven ACS patients (acute myocardial infarction and unstable angina and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA and chest pain syndrome (CPS. The frequencies of circulating CD4(+LAP(+ Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP on CD4(+ T cells were determined by flow cytometry. The function of CD4(+LAP(+ Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10 and transforming growth factor-β protein (TGF-β levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs was measured by real time-polymerase chain reaction. RESULTS: We found ACS patients had a significantly lower frequency of circulating CD4(+LAP(+ Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4(+ T cells and the serum levels of TGF-β in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. CONCLUSIONS: A novel regulatory T cell subset, defined as CD4(+LAP(+ T cells is defective in ACS patients.

  10. Clinical Applications of NanoVelcro Rare-Cell Assays for Detection and Characterization of Circulating Tumor Cells.

    Science.gov (United States)

    Chen, Jie-Fu; Zhu, Yazhen; Lu, Yi-Tsung; Hodara, Elisabeth; Hou, Shuang; Agopian, Vatche G; Tomlinson, James S; Posadas, Edwin M; Tseng, Hsian-Rong

    2016-01-01

    Liquid biopsy of tumor through isolation of circulating tumor cells (CTCs) allows non-invasive, repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic cancer, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e., NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detection isolation, and characterization of CTCs in peripheral blood, providing an opportunity to noninvasively monitor disease progression in individual cancer patients. Over the past decade, a series of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCs as a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expression profiling. In this review article, the authors will briefly introduce the development of three generations of NanoVelcro CTC Assays, and highlight the clinical applications of each generation for various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, and melanoma.

  11. Clinical utility of circulating tumor cell counting through CellSearch®: the dilemma of a concept suspended in Limbo

    Directory of Open Access Journals (Sweden)

    Raimondi C

    2014-04-01

    Full Text Available Cristina Raimondi,1 Angela Gradilone,1 Giuseppe Naso,2 Enrico Cortesi,2 Paola Gazzaniga1 1Dipartimento Medicina Molecolare, Sapienza Università di Roma, Rome, Italy; 2Dipartimento di Scienze Radiologiche, Oncologiche e Anatomopatologiche, Sapienza Università di Roma, Rome, Italy Abstract: To date, 10 years after the first demonstration of circulating tumor cells (CTCs, prognostic significance in metastatic breast cancer using the US Food and Drug Administration–cleared system CellSearch®, the potential utility of CTCs in early clinical development of drugs, their role as a surrogate marker of response to therapy, and their molecular analysis for patient stratification for targeted therapies are still major unsolved questions. Great expectations are pinned on the ongoing interventional trials aimed to demonstrate that CTCs might be of value for guiding treatment of patients and predicting cancer progression. To fill the gap between theory and practice with regard to the clinical utility of CTCs, a bridge is needed, taking into account innovative design for clinical trials, a revised definition of traditional CTCs, next-generation CTC technology, the potential clinical application of CTC analysis in non-validated settings of disease, and finally, expanding the number of patients enrolled in the studies. In this regard, the results of the first European pooled analysis definitely validated the independent prognostic value of CTC counting in metastatic breast cancer patients. Keywords: CTC, clinical trials, prognosis

  12. High-Throughput Selective Capture of Single Circulating Tumor Cells by Dielectrophoresis at a Wireless Electrode Array.

    Science.gov (United States)

    Li, Min; Anand, Robbyn K

    2017-07-05

    We demonstrate continuous high-throughput selective capture of circulating tumor cells by dielectrophoresis at arrays of wireless electrodes (bipolar electrodes, BPEs). The use of BPEs removes the requirement of ohmic contact to individual array elements, thus enabling otherwise unattainable device formats. Capacitive charging of the electrical double layer at opposing ends of each BPE allows an AC electric field to be transmitted across the entire device. Here, two such designs are described and evaluated. In the first design, BPEs interconnect parallel microchannels. Pockets extruding from either side of the microchannels volumetrically control the number of cells captured at each BPE tip and enhance trapping. High-fidelity single-cell capture was achieved when the pocket dimensions were matched to those of the cells. A second, open design allows many non-targeted cells to pass through. These devices enable high-throughput capture of rare cells and single-cell analysis.

  13. Intensive chemotherapy for acute myeloid leukemia differentially affects circulating TC1, TH1, TH17 and TREG cells

    Directory of Open Access Journals (Sweden)

    Gjertsen Bjørn

    2010-07-01

    Full Text Available Abstract Background Several observations suggest that immunological events early after chemotherapy, possibly during the period of severe treatment-induced cytopenia, are important for antileukemic immune reactivity in acute myeloid leukemia (AML. We therefore investigated the frequencies of various T cell subsets (TC1, TH1, TH17 and CD25+ FoxP3+ TREG cells in AML patients with untreated disease and following intensive chemotherapy. Results Relative levels of circulating TC1 and TH1 cells were decreased in patients with severe chemotherapy-induced cytopenia, whereas TH17 levels did not differ from healthy controls. Increased levels of regulatory CD25+ FoxP3+ T cells were detected in AML patients with untreated disease, during chemotherapy-induced cytopenia and during regeneration after treatment. TH17 and TH1 levels were significantly higher in healthy males than females, but this gender difference was not detected during chemotherapy-induced cytopenia. Finally, exogenous IL17-A usually had no or only minor effects on proliferation of primary human AML cells. Conclusions We conclude that the effect of intensive AML chemotherapy differ between circulating T cell subsets, relative frequencies of TH17 cells are not affected by chemotherapy and this subset may affect AML cells indirectly through their immunoregulatory effects but probably not through direct effects of IL17-A.

  14. Circulating blocking factors of lymphoid-cell cytotoxicity in x-ray-induced rat small-bowel adenocarcinoma

    International Nuclear Information System (INIS)

    Stevens, R.H.; Brooks, G.P.; Osborne, J.W.

    1979-01-01

    Circulating blocking factors capable of abrogating cell-mediated immune responses measured by in vitro lymphoid-cell cytotoxicity were identified in the sera of Holtzman outbred rats 6 to 9 months after a single exposure of only the temporarily exteriorized, hypoxic ileum and jejunum to 1700 to 2000 R of X radiation. Such factors were found to exist in the serum of every animal exposed to the ionizing radiation regardless of whether a visibly identifiable small-bowel adenocarcinoma existed or subsequently would develop. Protection of cultured x-ray-induced rat small-bowel cancer cells from destruction by tumor-sensitized lymphoid cells as measured by the release of lactoperoxidase-catalyzed radioiodinated membrane proteins from the tumor target cells was conferred by the action of the blocking factors at both effector and target cell levels. The results of this study demonstrate that exposure of only the rat small intestine to ionizing radiation leads to elaboration of circulating factors identifiable several months postirradiation which will block cell-mediated immune responses directed against cancer cells developing in the exposed tissue

  15. A higher frequency of circulating IL-22(+CD4(+ T cells in Chinese patients with newly diagnosed Hashimoto's thyroiditis.

    Directory of Open Access Journals (Sweden)

    Hui Guo

    Full Text Available BACKGROUND: IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22(+ and IL-17A(+ CD4(+ T cells in the pathogenesis of Hashimoto's thyroiditis (HT is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22(+ CD4(+ T cells in HT patients to understand their roles in the pathogenesis of HT. METHODS: The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22(+CD4(+ and IL-17A(+CD4(+ T cells in 17 HT patients and 17 healthy controls (HC were determined by enzyme-linked immunosorbent assay (ELISA and flow cytometry. The levels of serum free triiodothyronine (FT4, free thyroxine (FT3, thyroid stimulating hormone (TSH, anti-thyroid peroxidase (TPO and anti-thyroglobulin antibodies (TgAb by chemiluminescent enzyme immunoassay and radioimmunoassay. RESULTS: The percentages of circulating IL-22(+CD4(+ and IL-17(+CD4(+ T cells (p<0.0001, p<0.0001 and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210 in the HT patients were significantly higher than that in the HC. The percentages of IL-22(+CD4(+ T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001 and IL-17A(+IL-22(+CD4(+ T cells (r = 0.8914, p<0.0001, but were negatively correlated with Th1 cells (r = -0.6110, p<0.0092 in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A(+IL-22(+CD4(+ T cells were negatively correlated with the levels of serum TSH in the HT patients (r = -0.8402, p<0.0001; r = -0.8589, p<0.0001; r = -0.8289 p<0.0001, respectively. CONCLUSIONS: A higher frequency of circulating IL-22(+CD4(+ and IL-17A(+CD4(+ T cells may be associated with the development of HT in Chinese patients.

  16. Pressure Regulator With Internal Ejector Circulation Pump, Flow and Pressure Measurement Porting, and Fuel Cell System Integration Options

    Science.gov (United States)

    Vasquez, Arturo

    2011-01-01

    An advanced reactant pressure regulator with an internal ejector reactant circulation pump has been developed to support NASA's future fuel cell power systems needs. These needs include reliable and safe operation in variable-gravity environments, and for exploration activities with both manned and un manned vehicles. This product was developed for use in Proton Exchange Membrane Fuel Cell (PEMFC) power plant reactant circulation systems, but the design could also be applied to other fuel cell system types, (e.g., solid-oxide or alkaline) or for other gas pressure regulation and circulation needs. The regulator design includes porting for measurement of flow and pressure at key points in the system, and also includes several fuel cell system integration options. NASA has recognized ejectors as a viable alternative to mechanical pumps for use in spacecraft fuel cell power systems. The ejector motive force is provided by a variable, high-pressure supply gas that travels through the ejector s jet nozzle, whereby the pressure energy of the fluid stream is converted to kinetic energy in the gas jet. The ejector can produce circulation-to-consumption-flow ratios that are relatively high (2-3 times), and this phenomenon can potentially (with proper consideration of the remainder of the fuel cell system s design) be used to provide completely for reactant pre-humidification and product water removal in a fuel cell system. Specifically, a custom pressure regulator has been developed that includes: (1) an ejector reactant circulation pump (with interchangeable jet nozzles and mixer sections, gas-tight sliding and static seals in required locations, and internal fluid porting for pressure-sensing at the regulator's control elements) and (2) internal fluid porting to allow for flow rate and system pressure measurements. The fluid porting also allows for inclusion of purge, relief, and vacuum-breaker check valves on the regulator assembly. In addition, this regulator could also

  17. What Drives Saline Circulation Cells in Coastal Aquifers? An Energy Balance for Density-Driven Groundwater Systems

    Science.gov (United States)

    Harvey, C. F.; Michael, H. A.

    2017-12-01

    We formulate the energy balance for coastal groundwater systems and apply it to: (1) Explain the energy driving offshore saline circulation cells, and; (2) Assess the accuracy of numerical simulations of coastal groundwater systems. The flow of fresh groundwater to the ocean is driven by the loss of potential energy as groundwater drops from the elevation of the inland watertable, where recharge occurs, to discharge at sea level. This freshwater flow creates an underlying circulation cell of seawater, drawn into coastal aquifers offshore and discharging near shore, that adds to total submarine groundwater discharge. The saline water in the circulation cell enters and exits the aquifer through the sea floor at the same hydraulic potential. Existing theory explains that the saline circulation cell is driven by mixing of fresh and saline without any additional source of potential or mechanical power. This explanation raises a basic thermodynamic question: what is the source of energy that drives the saline circulation cell? Here, we resolve this question by building upon Hubbert's conception of hydraulic potential to formulate an energy balance for density-dependent flow and salt transport through an aquifer. We show that, because local energy dissipation within the aquifer is proportional to the square of the groundwater velocity, more groundwater flow may be driven through an aquifer for a given energy input if local variations in velocity are smoothed. Our numerical simulations of coastal groundwater systems show that dispersion of salt across the fresh-saline interface spreads flow over larger volumes of the aquifer, smoothing the velocity field, and increasing total flow and submarine groundwater discharge without consuming more power. The energy balance also provides a criterion, in addition to conventional mass balances, for judging the accuracy of numerical solutions of non-linear density-dependent flow problems. Our results show that some numerical

  18. Optimization of therapeutic proteins to delete T-cell epitopes while maintaining beneficial residue interactions.

    Science.gov (United States)

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-04-01

    Exogenous enzymes, signaling peptides, and other classes of nonhuman proteins represent a potentially massive but largely untapped pool of biotherapeutic agents. Adapting a foreign protein for therapeutic use poses numerous design challenges. We focus here on one significant problem: modifying the protein to mitigate the immune response mounted against "non-self" proteins, while not adversely affecting the protein's stability or therapeutic activity. In order to propose such variants suitable for experimental evaluation, this paper develops a computational method to select sets of mutations predicted to delete immunogenic T-cell epitopes, as evaluated by a 9-mer potential, while simultaneously maintaining important residues and residue interactions, as evaluated by one- and two-body potentials. While this design problem is NP-hard, we develop an integer programming approach that works very well in practice. We demonstrate the effectiveness of our approach by developing plans for biotherapeutic proteins that, in previous studies, have been partially deimmunized via extensive experimental characterization and modification of limited segments. In contrast, our global optimization technique considers an entire protein and accounts for all residues, residue interactions, and epitopes in proposing candidates worth subjecting to experimental evaluation.

  19. Effects of TiO₂ and Co₃O₄ nanoparticles on circulating angiogenic cells.

    Directory of Open Access Journals (Sweden)

    Valentina Spigoni

    Full Text Available Sparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs and cardiovascular (CV risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO2 and Co3O4 NPs in human circulating angiogenic cells (CACs, which take part in vascular endothelium repair/replacement.CACs were isolated from healthy donors' buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO2 and Co3O4 (from 1 to 100 μg/ml to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation, function (fibronectin adhesion assay, oxidative stress and inflammatory cytokine gene expression.Neither oxidative stress nor cell death were associated with exposure to TiO2 NP (except at the highest concentration tested, which, however, induced a higher pro-inflammatory effect compared to Co3O4 NPs (p<0.01. Exposure to Co3O4 NPs significantly reduced cell viability (p<0.01 and increased caspase activity (p<0.01, lipid peroxidation end-products (p<0.05 and pro-inflammatory cytokine gene expression (p<0.05 or lower. Notably, CAC functional activity was impaired after exposure to both TiO2 (p<0.05 or lower and Co3O4 (p<0.01 NPs.In vitro exposure to TiO2 and Co3O4 NPs exerts detrimental effects on CAC viability and function, possibly mediated by accelerated apoptosis, increased oxidant stress (Co3O4 NPs only and enhancement of inflammatory pathways (both TiO2 and Co3O4 NPs. Such adverse effects may be relevant for a potential role of exposure to TiO2 and Co3O4 NPs in enhancing CV risk in humans.

  20. Effects of TiO2 and Co3O4 Nanoparticles on Circulating Angiogenic Cells

    Science.gov (United States)

    Spigoni, Valentina; Cito, Monia; Alinovi, Rossella; Pinelli, Silvana; Passeri, Giovanni; Zavaroni, Ivana; Goldoni, Matteo; Campanini, Marco; Aliatis, Irene; Mutti, Antonio

    2015-01-01

    Background and Aim Sparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs) and cardiovascular (CV) risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO2 and Co3O4 NPs in human circulating angiogenic cells (CACs), which take part in vascular endothelium repair/replacement. Methods CACs were isolated from healthy donors’ buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO2 and Co3O4 (from 1 to 100 μg/ml) to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation), function (fibronectin adhesion assay), oxidative stress and inflammatory cytokine gene expression. Results Neither oxidative stress nor cell death were associated with exposure to TiO2 NP (except at the highest concentration tested), which, however, induced a higher pro-inflammatory effect compared to Co3O4 NPs (p<0.01). Exposure to Co3O4 NPs significantly reduced cell viability (p<0.01) and increased caspase activity (p<0.01), lipid peroxidation end-products (p<0.05) and pro-inflammatory cytokine gene expression (p<0.05 or lower). Notably, CAC functional activity was impaired after exposure to both TiO2 (p<0.05 or lower) and Co3O4 (p<0.01) NPs. Conclusions In vitro exposure to TiO2 and Co3O4 NPs exerts detrimental effects on CAC viability and function, possibly mediated by accelerated apoptosis, increased oxidant stress (Co3O4 NPs only) and enhancement of inflammatory pathways (both TiO2 and Co3O4 NPs). Such adverse effects may be relevant for a potential role of exposure to TiO2 and Co3O4 NPs in enhancing CV risk in humans. PMID:25803285

  1. Circulating progenitor cell count for cardiovascular risk stratification: a pooled analysis.

    Directory of Open Access Journals (Sweden)

    Gian Paolo Fadini

    Full Text Available BACKGROUND: Circulating progenitor cells (CPC contribute to the homeostasis of the vessel wall, and a reduced CPC count predicts cardiovascular morbidity and mortality. We tested the hypothesis that CPC count improves cardiovascular risk stratification and that this is modulated by low-grade inflammation. METHODOLOGY/PRINCIPAL FINDINGS: We pooled data from 4 longitudinal studies, including a total of 1,057 patients having CPC determined and major adverse cardiovascular events (MACE collected. We recorded cardiovascular risk factors and high-sensitive C-reactive protein (hsCRP level. Risk estimates were derived from Cox proportional hazard analyses. CPC count and/or hsCRP level were added to a reference model including age, sex, cardiovascular risk factors, prevalent CVD, chronic renal failure (CRF and medications. The sample was composed of high-risk individuals, as 76.3% had prevalent CVD and 31.6% had CRF. There were 331 (31.3% incident MACE during an average 1.7+/-1.1 year follow-up time. CPC count was independently associated with incident MACE even after correction for hsCRP. According to C-statistics, models including CPC yielded a non-significant improvement in accuracy of MACE prediction. However, the integrated discrimination improvement index (IDI showed better performance of models including CPC compared to the reference model and models including hsCRP in identifying MACE. CPC count also yielded significant net reclassification improvements (NRI for CV death, non-fatal AMI and other CV events. The effect of CPC was independent of hsCRP, but there was a significant more-than-additive interaction between low CPC count and raised hsCRP level in predicting incident MACE. CONCLUSIONS/SIGNIFICANCE: In high risk individuals, a reduced CPC count helps identifying more patients at higher risk of MACE over the short term, especially in combination with a raised hsCRP level.

  2. Inhibition of circulating immune cell activation: a molecular antiinflammatory effect of the Mediterranean diet.

    Science.gov (United States)

    Mena, Mari-Pau; Sacanella, Emilio; Vazquez-Agell, Mónica; Morales, Mercedes; Fitó, Montserrat; Escoda, Rosa; Serrano-Martínez, Manuel; Salas-Salvadó, Jordi; Benages, Neus; Casas, Rosa; Lamuela-Raventós, Rosa M; Masanes, Ferran; Ros, Emilio; Estruch, Ramon

    2009-01-01

    Adherence to the Mediterranean diet (Med-Diet) is associated with a reduced risk of cardiovascular disease (CVD). However, the molecular mechanisms involved are not fully understood. The objective was to compare the effects of 2 Med-Diets with those of a low-fat diet on immune cell activation and soluble inflammatory biomarkers related to atherogenesis in subjects at high risk of CVD. In a controlled study, we randomly assigned 112 older subjects with diabetes or > or =3 CVD risk factors to 3 dietary intervention groups: Med-Diet with supplemental virgin olive oil (VOO), Med-Diet with supplemental nuts, and low-fat diet. Changes from baseline in cellular and serum inflammatory biomarkers were assessed at 3 mo. One hundred six participants (43% women; average age: 68 y) completed the study. At 3 mo, monocyte expression of CD49d, an adhesion molecule crucial for leukocyte homing, and of CD40, a proinflammatory ligand, decreased (P < 0.05) after both Med-Diets but not after the low-fat diet. Serum interleukin-6 and soluble intercellular adhesion molecule-1, inflammatory mediators crucial in firm adhesion of leukocytes to endothelial surfaces, decreased (P < 0.05) in both Med-Diet groups. Soluble vascular cellular adhesion molecule-1 and C-reactive protein decreased only after the Med-Diet with VOO (P < 0.05), whereas interleukin-6, soluble vascular cellular adhesion molecule-1, and soluble intercellular adhesion molecule-1 increased (P < 0.05) after the low-fat diet. Med-Diets supplemented with VOO or nuts down-regulate cellular and circulating inflammatory biomarkers related to atherogenesis in subjects at high risk of CVD. The results support the recommendation of the Med-Diet as a useful tool against CVD.

  3. Clinical utility of circulating cell-free DNA in advanced colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Allan A Lima Pereira

    Full Text Available Circulating cell-free DNA (cfDNA isolated from the plasma of cancer patients (pts has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC has not been previously described.Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC], with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility.128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128 of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30 of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents.cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.

  4. Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA.

    Science.gov (United States)

    Worm Ørntoft, Mai-Britt; Jensen, Sarah Østrup; Hansen, Thomas Birkballe; Bramsen, Jesper Bertram; Andersen, Claus Lindbjerg

    2017-08-01

    Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. To compare cfDNA recovery of different BSC methods, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only 3 kits recovered DNA fragments of BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

  5. Expansion of Circulating T Follicular Helper Cells in Children with Acute Henoch-Schönlein Purpura

    Science.gov (United States)

    Xie, Jue; Liu, Yan; Wang, Lei; Ruan, Guoxiang; Yuan, Huiming; Fang, Hong; Wu, Jianyong; Cui, Dawei

    2015-01-01

    Henoch-Schönlein purpura (HSP) is a common systemic small vessel vasculitis in children with disorder autoimmune responses. T follicular helper (TFH) cells play crucial roles in regulating immune responses. The aim of our study was to investigate the probable role of TFH cells in the pathogenesis of children with HSP. In this study, the frequency of circulating CXCR5+CD4+TFH cells with inducible costimulator (ICOS) expression in the children with acute HSP was significantly higher than that in healthy controls (HCs) but not CXCR5+CD4+TFH cells with programmed death-1 (PD-1) expression. Moreover, serum levels of IL-21 and IL-6 cytokines, IgA, and C3 in HSP children were also significantly higher than those in HCs. A positive correlation was observed between the frequencies of circulating ICOS+CXCR5+CD4+TFH cells and the serum IL-21 or IgA levels of acute HSP children, respectively. Additionally, the mRNA expression levels of interleukin- (IL-) 21, IL-6, and transcriptional factors (B-cell lymphoma-6, Bcl-6) were also significantly increased in peripheral blood from acute HSP children compared to HCs. Taken together, these findings suggest that TFH cells and associated molecules might play critical roles in the pathogenesis of HSP, which are possible therapeutic targets in HSP children. PMID:26491701

  6. Nanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model.

    Science.gov (United States)

    Muhanna, Nidal; Mepham, Adam; Mohamadi, Reza M; Chan, Harley; Khan, Tahsin; Akens, Margarete; Besant, Justin D; Irish, Jonathan; Kelley, Shana O

    2015-10-01

    Circulating tumor cells (CTCs) can be used as markers for the detection, characterization, and targeted therapeutic management of cancer. We recently developed a nanoparticle-mediated approach for capture and sorting of CTCs based on their specific epithelial phenotype. In the current study, we investigate the phenotypic transition of tumor cells in an animal model and show the correlation of this transition with tumor progression. VX2 tumor cells were injected into rabbits, and CTCs were evaluated during tumor progression and correlated with computerized tomography (CT) measurements of tumor volume. The results showed a dramatic increase of CTCs during the four weeks of tumor growth. Following resection, CTC levels dropped but then rebounded, likely due to lymph node metastases. Additionally, CTCs showed a marked loss of the epithelial cell adhesion molecule (EpCAM) relative to precursor cells. In conclusion, the device accurately traces disease progression and CTC phenotypic shift in an animal model. The detection of circulating tumor cells (CTCs) has been used to predict disease prognosis. In this study, the authors developed a nanoparticle-mediated platform based on microfluidics to analyze the differential expressions of epithelial cell adhesion molecule (EpCAM) on CTCs in an animal model. It was found that the loss of EpCAM correlated with disease progression. Hence, the use of this platform may be further applied in other cancer models in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Circulating hematopoietic progenitors and CD34(+) cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution.

    Science.gov (United States)

    Yu, Jui-Ting; Cheng, Shao-Bin; Yang, Youngsen; Chang, Kuang-Hsi; Hwang, Wen-Li; Teng, Chieh-Lin Jerry

    2016-01-01

    Previous studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC) and CD34(+) cell are positively correlated with CD34(+) cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34(+) cells required for performing an efficient hematopoietic stem cell (HSC) harvest in lymphoma and myeloma patients have not been defined in our institution. Medical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×10(6) CD34(+) cells/kg and ≥5×10(6) CD34(+) cells/kg, respectively. The minimally required or optimal HSC yield obtained was not influenced by age (≥60 years), sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34(+) cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34(+) cell) and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34(+) cell). The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm(3) for HPCs and 10/mm(3) for CD34(+) cells. Furthermore, the cell count cutoff for obtaining optimal HSC harvest was determined to be 60/mm(3) for HPCs and 35/mm(3) for CD34(+) cells. A total of 60/mm(3) of HPCs and 35/mm(3) of CD34(+) cells in peripheral blood predicted optimal HSC harvest in lymphoma and myeloma patients.

  8. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  9. Combined analysis of circulating epithelial cells and serum thyroglobulin for distinguishing disease status of the patients with papillary thyroid carcinoma

    OpenAIRE

    Lin, Hung-Chih; Liou, Miaw-Jene; Hsu, Hsung-Ling; Hsieh, Jason Chia-Hsun; Chen, Yi-An; Tseng, Ching-Ping; Lin, Jen-Der

    2015-01-01

    Papillary thyroid carcinoma (PTC) accounts for about 80% of the cases in thyroid cancer. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. Whether enumeration of circulating epithelial cells (CECs) helps to define disease status of PTC patients was investigated. CECs were enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) ...

  10. In vitro validation of an ultra-sensitive scanning fluorescence microscope for analysis of Circulating Tumor Cells

    DEFF Research Database (Denmark)

    Hillig, Thore; Nygaard, Ann-Britt; Nekiunaite, Laura

    2014-01-01

    Analysis of circulating tumor cells (CTC) holds promise of providing liquid biopsies from patients with cancer. However, current methods include enrichment procedures. We present a method (CytoTrack), where CTC from 7.5 mL of blood is stained, analyzed and counted by a scanning fluorescence...... microscope. The method was validated by breast cancer cells (MCF-7) spiked in blood from healthy donors. The number of cells spiked in each blood sample was exactly determined by cell sorter and performed in three series of three samples spiked with 10, 33 or 100 cells in addition with three control samples...... detect breast cancer cells in spiking experiments and should be tested on blood samples from breast cancer patients. The method could benefit from automation that could reduce the CV%, and further optimization of the procedure to increase the recovery....

  11. Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia.

    Science.gov (United States)

    Rissiek, Anne; Schulze, Christian; Bacher, Ulrike; Schieferdecker, Aneta; Thiele, Benjamin; Jacholkowski, Anita; Flammiger, Anna; Horn, Christiane; Haag, Friedrich; Tiegs, Gisa; Zirlik, Katja; Trepel, Martin; Tolosa, Eva; Binder, Mascha

    2014-11-15

    Antitumor immunity in chronic lymphocytic leukemia (CLL) is hampered by highly dysfunctional T-cells. Although certain T-cell subsets have been reported to be of prognostic significance in this disease, their interplay is complex and it remains incompletely understood which of these subsets significantly drive CLL progression. Here, we determined immunological profiles of 24 circulating T-cell subsets from 79 untreated individuals by multiparametric flow cytometry. This screening cohort included healthy donors, patients with monoclonal B-cell lymphocytosis (MBL), Rai 0 CLL and advanced CLL. We applied multidimensional scaling analysis as rigorous and unbiased statistical tool to globally assess the composition of the circulating T-cell environment and to generate T-cell scores reflecting its integrity. These scores allowed clear distinction between advanced CLL and healthy controls, whereas both MBL and Rai 0 CLL showed intermediate scores mirroring the biological continuum of CLL and its precursor stages. T-cell stimulation and suppression assays as well as longitudinal T-cell profiling showed an increasingly suppressive regulatory function initiating at the MBL stage. Effector function was impaired only after transition to CLL and partially recovered after chemoimmunotherapy. In an independent validation cohort of 52 untreated CLL cases, aberrant T-cell profiles were significantly associated with shorter time to treatment independently of other prognostic parameters. Random forest modeling predicted regulatory T-cell, gamma/delta and NKT-cells, as well as exhaustion of the CD8+ subset as potential drivers of progression. Our data illustrate a pathological T-cell environment in MBL that evolves toward a more and more suppressive and prognostically relevant profile across the disease stages. © 2014 UICC.

  12. De Novo Circulating Antidonor's Cell Antibodies During Induced Acute Rejection of Allogeneic Myofibers in Myogenic Cell Transplantation: A Study in Nonhuman Primates

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    Daniel Skuk, MD

    2017-12-01

    Conclusions. Flow cytometry detection of de novo circulating antibodies against the donor’s cells was consistently associated with AR. A clear increase in this antibody detection indicated current or recent AR. Smaller increases in comparison to the preimmunosuppression values were not associated with AR.

  13. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Directory of Open Access Journals (Sweden)

    Goli eSamimi

    2015-04-01

    Full Text Available A range of molecular alterations found in tumor cells, such as DNA mutations and methylation changes, is also reflected in cell-free circulating DNA (circDNA released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. DNA methylation is a common molecular alteration found in many cancer types. Unlike DNA mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. DNA methylation is reflected within circDNA and therefore detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

  14. Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells.

    Science.gov (United States)

    Hensel, Michael T; Peng, Tao; Cheng, Anqi; De Rosa, Stephen C; Wald, Anna; Laing, Kerry J; Jing, Lichen; Dong, Lichun; Magaret, Amalia S; Koelle, David M

    2017-10-01

    Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation. IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents

  15. Experimental findings on thermal use of residues and biofuels in circulating fluidized bed combustion systems; Experimentelle Ergebnisse zur thermischen Nutzung von Rest- und Biobrennstoffen in zirkulierenden Wirbelschichtfeuerungen

    Energy Technology Data Exchange (ETDEWEB)

    Bernstein, W.; Brunne, T.; Girndt, H. [Technische Univ. Dresden (Germany); Albrecht, J. [Lurgi Lentjes Babcock, Frankfurt am Main (Germany); Youssef, M. [Minia Univ. (Egypt)

    1996-12-31

    The energy Engineering Institute of Dresden Technical University investigated the combustion and emission characteristics of a number of combustion systems, including a circulating fluidized bed system with a capacity of 0.3 MW{sub th}. Egypt`s sugar cane industry produces large volumes of bagasse. The conbustion and emission characteristics of this biofuel in a circulating fludized bed combustion systems were investigated in a joint research project of the University of Minia and Dresden Technical University. (orig.) [Deutsch] Am Institut fuer Energietechnik der TU Dresden wird das Verbrennungs- und Emissionsverhalten verschiedenster Brennstoffe in unterschiedlichen Feuerungssystemen untersucht. Neben anderen Pilotanlagen steht eine zirkulierende Wirbelschichtfeuerung (ZWFS) mit einer Leistung von 0.3 MW{sub th} zur Verfuegung. In der Zuckerrohrindustrie Aegyptens fallen grosse Mengen von Bagasse an. In einer gemeinsamen Forschungsarbeit zwischen der Universitaet Minia und der TU Dresden sollte das Verbrennungs- und Emissionsverhalten dieses Biobrennstoffes in einer ZWSF untersucht werden. (orig)

  16. Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children.

    Science.gov (United States)

    Nébor, Danitza; Romana, Marc; Santiago, Raoul; Vachiery, Nathalie; Picot, Julien; Broquere, Cédric; Chaar, Vicky; Doumdo, Lydia; Odièvre, Marie-Hélène; Benkerrou, Malika; Elion, Jacques

    2013-06-01

    Microparticles are cell membrane-derived microvesicles released during cell apoptosis and activation processes. They have been described as bio-markers in various vascular diseases, including sickle cell anemia, and associated with an increased risk of thrombosis. We investigated the effects of fetal hemoglobin level, a factor known to modulate the clinical expression of sickle cell anemia, and that of hydroxycarbamide treatment which reduces the frequency of vasoocclusive crises, the canonical clinical manifestation of the disease, on both the plasma concentration and the cellular origin of circulating microparticles. Flow cytometry was used to characterize microparticles in 62 sickle cell anemia children at steady state aged 2 months-16 years; 13 of them were treated with hydroxycarbamide. In untreated children, we observed negative correlations between fetal hemoglobin levels and the absolute plasma concentration of microparticles as well as that of microparticles specifically derived from platelets, erythrocytes, and monocytes. Compared to untreated children, those treated with hydroxyurea showed lower concentrations of total microparticles as a consequence of decreased microparticles shed by platelets and erythrocytes. In conclusion, in our sickle cell patients, neonatal decline of fetal hemoglobin coincided with an increase in circulating microparticles derived from erythrocytes, platelets, and monocytes. Hydroxyurea treatment was associated with a decrease in microparticles derived from erythrocytes and platelets.

  17. The level of circulating endothelial progenitor cells may be associated with the occurrence and recurrence of chronic subdural hematoma

    Directory of Open Access Journals (Sweden)

    Yan Song

    2013-01-01

    Full Text Available OBJECTIVES: The onset of chronic subdural hematoma may be associated with direct or indirect minor injuries to the head or a poorly repaired vascular injury. Endothelial progenitor cells happen to be one of the key factors involved in hemostasis and vascular repair. This study was designed to observe the levels of endothelial progenitor cells, white blood cells, platelets, and other indicators in the peripheral blood of patients diagnosed with chronic subdural hematoma to determine the possible relationship between the endothelial progenitor cells and the occurrence, development, and outcomes of chronic subdural hematoma. METHOD: We enrolled 30 patients with diagnosed chronic subdural hematoma by computer tomography scanning and operating procedure at Tianjin Medical University General Hospital from July 2009 to July 2011. Meanwhile, we collected 30 cases of peripheral blood samples from healthy volunteers over the age of 50. Approximately 2 ml of blood was taken from veins of the elbow to test the peripheral blood routine and coagulation function. The content of endothelial progenitor cells in peripheral blood mononuclear cells was determined by flow cytometry. RESULTS: The level of endothelial progenitor cells in peripheral blood was significantly lower in preoperational patients with chronic subdural hematomas than in controls. There were no significant differences between the two groups regarding the blood routine and coagulation function. However, the levels of circulating endothelial progenitor cells were significantly different between the recurrent group and the non-recurrent group. CONCLUSIONS: The level of circulating endothelial progenitor cells in chronic subdural hematoma patients was significantly lower than the level in healthy controls. Meanwhile, the level of endothelial progenitor cells in recurrent patients was significantly lower than the level in patients without recurrence. Endothelial progenitor cells may be related to the

  18. Increased circulating follicular regulatory T cells are associated with lower auto-antibodies in stable remission rheumatoid arthritis patients.

    Science.gov (United States)

    Liu, Chen; Wang, Dongwei; Lu, Songsong; Xu, Qinzhu; Zhao, Lei; Zhao, Jingzhong; Song, Ying; Wang, Hui

    2018-01-30

    To examine expression and function changes of circulating CD4 + CXCR5 + FoxP3 + Follicular regulatory T (TFR) cells in active and stable remission rheumatoid arthritis (RA) patients and to clarify the role of TFR cells in the pathogenesis of RA. TFR and follicular helper T cells (TFH) levels in peripheral blood of 39 active RA patients, 39 stable remission RA patients and 33 healthy controls were detected by flow cytometry. The function of TFR cells was measured by co-culturing with TFH and B cells. The activated CD45RA - FoxP3 hi TFR cells were analyzed as well. Clinical indicators including serum levels of Ig and auto-antibodies were tested and correlations with TFR cells were systematically analyzed. DAS28 scores were further calculated and correlation analysis with TFR was conducted as well. The level of CD4 + CXCR5 + FOXP3 + TFR cells and the ratio of TFR/TFH in peripheral blood of stable remission RA patients were significantly increased compared with active RA patients and healthy controls. The function of TFR was enhanced and the activated CD45RA - FoxP3 hi TFR subset was increased in stable remission RA patients compared with healthy controls. Furthermore, TFR cells in RA patients were inversely correlated with Ig G, RF, anti-CCP, as well as DAS28 scores. Circulating TFR cells were increased as RA patients develop to a stable state and increased TFR cells could suppress autoimmunity of RA patients to stabilize their condition. Our results provide novel insight into the pathogenesis process of RA. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Islet autoantibodies and residual beta cell function in type 1 diabetes children followed for 3-6 years

    DEFF Research Database (Denmark)

    Sørensen, Jesper Sand; Vaziri-Sani, Fariba; Maziarz, M

    2012-01-01

    To test if islet autoantibodies at diagnosis of type 1 diabetes (T1DM) and after 3-6 years with T1D predict residual beta-cell function (RBF) after 3-6 years with T1D.......To test if islet autoantibodies at diagnosis of type 1 diabetes (T1DM) and after 3-6 years with T1D predict residual beta-cell function (RBF) after 3-6 years with T1D....

  20. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice

    DEFF Research Database (Denmark)

    Bentzon, Jacob Fog; Weile, Charlotte; Sondergaard, Claus S

    2006-01-01

    Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE-/-) mice have concluded that a substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of hematopoietic origin. This pathway, however, remains controversial. In the present...... study, we reexamined the origin of plaque SMCs in apoE-/- mice by a series of BM transplantations and in a novel model of atherosclerosis induced in surgically transferred arterial segments....

  1. Effects of bone marrow stromal cells and umbilical cord blood-derived stromal cells on daunorubicin-resistant residual Jurkat cells.

    Science.gov (United States)

    Liang, X; Hao, L; Chen, X; Zhang, X; Kong, P; Peng, X; Gao, L; Zhang, C; Wang, Q

    2010-11-01

    To observe the effects of the hematopoietic inductive microenvironment (HIM) simulated by stromal cells of different origins on daunorubicin-resistant residual Jurkat cells (Jurkat/DNR cells). Jurkat/DNR cells were cultured and identified. Human umbilical cord blood-derived stromal cells (UCBDSCs) and normal human bone marrow stromal cells (BMSCs) were isolated and cocultured with Jurkat/DNR cells. Jurkat/DNR cells were collected after 14 days of coculture and analyzed with regard to cell proliferation and differentiation abilities, apoptosis, drug sensitivity, and MRD1 multidrug resistance gene mRNA expression. UCBDSC-simulated HIM suppressed proliferation and promoted apoptosis, differentiation, and drug sensitivity of Jurkat/DNR cells more significantly than BMSC-simulated HIM. Both BMSCs and UCBDSCs reconstruct the leukemic HIM and reverse drug resistance in Jurkat/DNR cells. UCBDSCs reconstruct the leukemic HIM and reverse drug resistance more significantly than BMSCs. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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 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.

  3. Circulating Tumor Cells Predict Occult Metastatic Disease and Prognosis in Pancreatic Cancer.

    Science.gov (United States)

    Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Winograd, Paul; Hou, Shuang; Song, Min; Wainberg, Zev A; Girgis, Mark D; Graeber, Thomas G; Agopian, Vatche G; Tseng, Hsian-Rong; Tomlinson, James S

    2018-04-01

    Occult metastatic tumors, below imaging thresholds, are a limitation of staging systems that rely on cross-sectional imaging alone and are a cause of the routine understaging of pancreatic ductal adenocarcinomas (PDACs). We investigated circulating tumor cells (CTCs) as a preoperative predictor of occult metastatic disease and as a prognostic biomarker for PDAC patients. A total of 126 patients (100 with cancer, 26 with benign disease) were enrolled in our study and CTCs were identified and enumerated from 4 mL of venous blood using the microfluidic NanoVelcro assay. CTC enumeration was correlated with clinicopathologic variables and outcomes following both surgical and systemic therapies. CTCs were identified in 78% of PDAC patients and CTC counts correlated with increasing stage (ρ = 0.42, p < 0.001). Of the 53 patients taken for potentially curative surgery, 13 (24.5%) had occult metastatic disease intraoperatively. Patients with occult disease had significantly more CTCs than patients with local disease only (median 7 vs. 1 CTC, p < 0.0001). At a cut-off of three or more CTCs/4 mL, CTCs correctly identified patients with occult metastatic disease preoperatively (area under the receiver operating characteristic curve 0.82, 95% confidence interval (CI) 0.76-0.98, p < 0.0001). CTCs were a univariate predictor of recurrence-free survival following surgery [hazard ratio (HR) 2.36, 95% CI 1.17-4.78, p = 0.017], as well as an independent predictor of overall survival on multivariate analysis (HR 1.38, 95% CI 1.01-1.88, p = 0.040). CTCs show promise as a prognostic biomarker for PDAC patients at all stages of disease being treated both medically and surgically. Furthermore, CTCs demonstrate potential as a preoperative biomarker for identifying patients at high risk of occult metastatic disease.

  4. Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

    Science.gov (United States)

    Soh, Kah Teong; Tario, Joseph D.; Wallace, Paul K.

    2018-01-01

    Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed. PMID:29128071

  5. Smoking reduces circulating CD26hi CD161hi MAIT cells in healthy individuals and patients with multiple sclerosis

    DEFF Research Database (Denmark)

    Ammitzbøll, Cecilie; Börnsen, Lars; Romme Christensen, Jeppe

    2017-01-01

    Upon chronic cigarette smoke exposure, inhaled antigens and irritants cause altered lung immune homeostasis. Circulating immune cells are affected, and smoking is associated with an increased risk of developing various disorders, including multiple sclerosis (MS). This study was conducted...

  6. Does Every Cell Get Blood? Young Students' Discussions about Illustrations of Human Blood Circulation

    Science.gov (United States)

    Westman, Anna-Karin; Karlsson, Karl-Goran

    2016-01-01

    This article presents a study of how groups of young students discuss illustrations of human blood circulation. Transparency is not an innate quality of illustrations, visual information is always coded and interpretations are always related to culture and context. Results of this study are discussed with reference to Kress and van Leeuwens'…

  7. Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Zhanshan [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Qian, Guangfang [Department of Endocrinology, Zhangqiu Municipal Hospital of Traditional Chinese Medicine, Zhangqiu, Shandong 250200 (China); Zang, Yan; Gu, Haihui; Huang, Yanyan; Zhu, Lishuang; Li, Jinqi; Liu, Yang; Tu, Xiaohua [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Song, Haihan [Emergency Center, East Hospital, Shanghai 200120 (China); Qian, Baohua, E-mail: qianbhl963@163.com [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China)

    2017-01-01

    Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5{sup +} CD4{sup +} T cells, in DLBCL. Data showed that compared to CXCR5{sup -} CD4{sup +} T cells, CXCR5{sup +} CD4{sup +} T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5{sup +} CD4{sup +} T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5{sup -} CD4{sup +} T cells, while the level of IL-10 secretion was significant elevated in the CXCR5{sup +} compartment compared to the CXCR5{sup -} compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5{sup +} CD4{sup +} T cell coculture compromised the CXCR5{sup +} CD4{sup +} T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5{sup +} compartment also contained significantly lower frequencies of cytotoxic CD4{sup +} T cells than the CXCR5{sup -} compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4{sup +} T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10. - Highlights: • We studied the role of the peripheral Tfh in DLBCL. • Tfh were effective at promoting the proliferation of primary DLBCL tumor cells. • Tfh were effective at inhibiting the apoptosis of primary DLBCL tumor cells. • IL-10 secretion in Tfh was significant elevated in DLBCL. • Neutralization of IL-10 compromised Tfh-mediated pro-tumor effects.

  8. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    International Nuclear Information System (INIS)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-01-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111 In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity

  9. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    Energy Technology Data Exchange (ETDEWEB)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

  10. Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells.

    Directory of Open Access Journals (Sweden)

    Dongfeng Qu

    Full Text Available Doublecortin-like kinase 1 (DCLK1 is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II compared to healthy volunteers (normal controls. No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT. Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.

  11. Circulating Prostate Cells Found in Men with Benign Prostate Disease Are P504S Negative: Clinical Implications

    Directory of Open Access Journals (Sweden)

    Nigel P. Murray

    2013-01-01

    Full Text Available Introduction. Developments in immunological and quantitative real-time PCR-based analysis have enabled the detection, enumeration, and characterization of circulating tumor cells (CTCs. It is assumed that the detection of CTCs is associated with cancer, based on the finding that CTCs can be detected in all major cancer and not in healthy subjects or those with benign disease. Methods and Patients. Consecutive men, with suspicion of prostate cancer, had blood samples taken before prostate biopsy; mononuclear cells were obtained using differential gel centrifugation and CPCs detecting using anti-PSA immunocytochemistry. Positive samples underwent further classification with anti-P504S. Results. 329 men underwent prostate biopsy; of these men 83 underwent a second biopsy and 44 a third one. Of those with a biopsy negative for cancer, 19/226 (8.4% had CPCs PSA (+ P504S (− detected at first biopsy, 6/74 (8.1% at second biopsy, and 5/33 (15.2% at third biopsy. Men with cancer-positive biopsies did not have PSA (+ P504S (− CPCs detected. These benign cells were associated with chronic prostatitis. Conclusions. Patients with chronic prostatitis may have circulating prostate cells detected in blood, which do not express the enzyme P504S and should be thought of as benign in nature.

  12. Polyethylene Glycol-Functionalized Magnetic Fe₃O₄/P(MMA-AA) Composite Nanoparticles Enhancing Efficient Capture of Circulating Tumor Cells.

    Science.gov (United States)

    Ma, Shaohua; Zhan, Xiaohui; Yang, Minggang; Lan, Fang; Wu, Yao; Gu, Zhongwei

    2018-04-01

    Circulating tumor cells (CTCs) played a significant role in early diagnosis and prognosis of carcinomas, and efficient capture of CTCs was highly desired to provide important and reliable evidence for clinical diagnosis. In present work, we successfully synthesized functional magnetic Fe3O4/P(MMA-AA) composite nanoparticles (FCNPs) inspired by a counterbalance concept for recognition and capture of CTCs. This counterbalance, composed of polyethylene glycol (PEG) suppressing cell adhesion and anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody targeting tumor cells, could both enhance the specific capture of tumor cells and reduce unspecific adhesion of normal cells. The study showed that the PEG density on the surface of the FCNPs affected the specificity of the materials, and a density of ca. 15% was efficient for reducing the unspecific adhesion. After incubation with the mixture of HepG2 cells and Jurkat T cells, the FCNPs reached a capture efficiency as high as about 86.5% of the cancer cells, suggesting great potential on detection of CTCs in the diagnoses and prognoses of cancer metastasis.

  13. Circulating hematopoietic progenitors and CD34+ cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution

    Directory of Open Access Journals (Sweden)

    Yu JT

    2016-02-01

    Full Text Available Jui-Ting Yu,1,2,* Shao-Bin Cheng,3,* Youngsen Yang,1 Kuang-Hsi Chang,4 Wen-Li Hwang,1 Chieh-Lin Jerry Teng,1,5,6 1Division of Hematology/Medical Oncology, Department of Medicine, Taichung Veterans General Hospital, 2Division of Hematology/Medical Oncology, Tungs' Taichung MetroHarbor Hospital, 3Division of General Surgery, Department of Surgery, 4Department of Medical Research and Education, Taichung Veterans General Hospital, 5Department of Life Science, Tunghai University, 6School of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China *These authors contributed equally to this work Background: Previous studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC and CD34+ cell are positively correlated with CD34+ cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34+ cells required for performing an efficient hematopoietic stem cell (HSC harvest in lymphoma and myeloma patients have not been defined in our institution. Patients and methods: Medical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×106 CD34+ cells/kg and ≥5×106 CD34+ cells/kg, respectively. Results: The minimally required or optimal HSC yield obtained was not influenced by age (≥60 years, sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34+ cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34+ cell and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34+ cell. The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm3 for HPCs and 10/mm3 for CD34+ cells. Furthermore, the cell count cutoff for obtaining

  14. Change in number and size of circulating tumor cells with high telomerase activity during treatment of patients with gastric cancer.

    Science.gov (United States)

    Ito, Hiroaki; Yamaguchi, Noriko; Onimaru, Manabu; Kimura, Satoshi; Ohmori, Tohru; Ishikawa, Fumihiro; Sato, Jun; Ito, Shun; Inoue, Haruhiro

    2016-12-01

    Detection of circulating tumor cells (CTCs) in peripheral blood is useful for estimating the prognosis of patients with cancer. We previously reported the detection of CTCs by OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. We demonstrated that the number of large (L)-GFP+ cells (≥7.735 µm in diameter) in peripheral blood samples correlated significantly with the prognosis of treatment-naïve gastric cancer patients, whereas the number of small (S)-GFP+ cells (number of GFP+ cells during treatment, and analyzed the association between the number of GFP+ cells in blood samples and the outcome of patients. Peripheral blood samples were obtained from 37 gastric patients prior and subsequent to surgery (three samples per time point). Upon infection of blood cells with OBP-401, GFP+ cells of different sizes were counted and measured. The association between the number of GFP+ cells and surgical outcome was determined by statistical analysis. The median follow-up period after surgery was 39 months. Although the difference was not significant, patients with ≥6 L-GFP+ cells in preoperative blood samples had a lower relapse-free survival rate than patients with 0-5 L-GFP+ cells. There was no significant correlation between the number of L-GFP+ cells in postoperative blood samples and the prognosis of patients receiving adjuvant therapy. Although the difference was not significant, the number of S-GFP+ cells in samples from patients who had received postoperative chemotherapy was higher than in those who had not. The number of L-GFP+ cells was not significantly correlated with the relapse-free survival rate in gastric cancer patients who underwent surgery. The number of S-GFP+ cells was relatively high in samples from patients who had received postoperative chemotherapy.

  15. Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: a prospective study.

    Science.gov (United States)

    Ito, Hiroaki; Inoue, Haruhiro; Kimura, Satoshi; Ohmori, Tohru; Ishikawa, Fumihiro; Gohda, Keigo; Sato, Jun

    2014-07-01

    The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

  16. Circulating, cell-free DNA as a marker for exercise load in intermittent sports

    OpenAIRE

    Haller, Nils; Helmig, Susanne; Taenny, Pascal; Petry, Julian; Schmidt, Sebastian; Simon, Perikles

    2018-01-01

    Background Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of ...

  17. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

    DEFF Research Database (Denmark)

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard

    2014-01-01

    BACKGROUND AND AIM: Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold...... of detection of a new generation BD FACSAria™ III digital flow cytometer. METHODS: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (

  18. Circulating regulatory Tfh cells are enriched in patients with chronic hepatitis B infection and induce the differentiation of regulatory B cells.

    Science.gov (United States)

    Wang, Rongxin; Xie, Ruiling; Song, Zongchang

    2018-04-15

    Chronic hepatitis B virus (HBV) infection is a complex disease with dysregulations in the immune system. Follicular helper T (Tfh) cells are professional B helper cells that are crucial to the development of antibody responses and are involved in a variety of diseases. In this study, we examined the circulating Tfh cells in patients with chronic HBV infection. We observed that CD3 + CD4 + CXCR5 + circulating Tfh cells contained a CD25 + Foxp3 + Treg-like subset that was significantly enriched in patients with chronic HBV infections. The CD25 + Tfh subset presented distinctive cytokine secretion profile, such as lower interferon (IFN)-γ and interleukin (IL)-17, and higher transforming growth factor (TGF)-β secretion, compared to the CD25 - Tfh subset. When incubated with autologous naive CD10 - CD27 - CD19 + B cells, the CD25 + Tfh subset was less capable of mediating CD20 -/lo CD38 + plasmablast differentiation than the CD25 - Tfh subset. In terms of Ig production, CD25 + Tfh cells were more potent at inducing IgM but less potent at inducing IgG and IgA than CD25 - Tfh cells. Interestingly, B cells following incubation with CD25 + Tfh cells presented elevated regulatory function, with higher production of IL-10 and enhanced capacity of suppressing autologous CD8 + T cell inflammation. In the chronic HBV-infected patients, the frequency of IL-10 + B cells and the HBV viral load were positively correlated with the frequency of CD25 + Foxp3 + CD4 + CXCR5 + Tfh cells. Together, this study presented that CD25 + Foxp3 + Treg-like Tfh cells were enriched in chronic HBV-infected patients and could promote regulatory B cell functions. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Combustion demonstration plant in circulant fluidized bed of residual coal; Planta de Demostracion de Combustion en Lecho Fluido Circulante de Carbones Residuales

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-12-31

    This report incorporates a summary of the operation results during the period of demonstration after started up. The report pretend to give an overview of the operation conditions along of the first year: Running hours, availability, electricity production, shooting downs, incidences, efficiency, fuel characteristics influence, pollutants emissions and standards comparations, etc. The main operation conclusions are: High availability, great number of running hours at full equivalent load; some months even over 100% regarding time scheduled. High reduction of gaseous emissions, really very low respecting the required by the applicable standards. It has been developed the engineering of a prototype project, by 30MW, using mixtures of solid fuels, residues and coals. (Author)

  20. Repeated bouts of exhaustive exercise increase circulating cell free nuclear and mitochondrial DNA without development of tolerance in healthy men.

    Science.gov (United States)

    Stawski, Robert; Walczak, Konrad; Kosielski, Piotr; Meissner, Pawel; Budlewski, Tomasz; Padula, Gianluca; Nowak, Dariusz

    2017-01-01

    Acute single strenuous exercise increases circulating cell free DNA (cf DNA). We tested whether three repeated bouts of exhaustive exercise induced the cf DNA response without development of tolerance in healthy men. Eleven average-trained men (age 34.0±5.2 years, body mass index 26.2±3.1 kg/m2, maximal oxygen consumption-VO2max 49.6±4.5 ml/kg*min) performed three treadmill exercise tests to exhaustion at speed corresponding to 70% VO2max separated by 72 hours of resting. Blood was collected before and after each bout of exercise for determination of cell free nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) by real-time PCR, selected markers of muscle damage, and blood cell count. Each bout induced the increase (pexercise, respectively. In a congruent way, cf mt-DNA rose significantly after the second (from 229±216 to 450±228*103 GE/mL) and third bout of exercise (from 173±120 to 462±314*103 GE/mL). Pre-exercise cf mt-DNA decreased (pexercise induced percentage increment of cf n-DNA was always many times higher than corresponding increments of the afore-mentioned markers at any occasion. Repeated bouts of exhaustive exercise induced remarkable increase in circulating cf n-DNA without signs of tolerance development. Baseline cf mt-DNA decreased in response to series of strenuous exercise. Since percentage increments of cf n-DNA in response to exercise were many times higher than those observed for other markers, measurement of circulating cf n-DNA could be a sensitive tool for monitoring acute exercise effects in human body.

  1. TLR activation excludes circulating naive CD8+ T cells from gut-associated lymphoid organs in mice.

    Science.gov (United States)

    Heidegger, Simon; Kirchner, Sophie-Kathrin; Stephan, Nicolas; Bohn, Bernadette; Suhartha, Nina; Hotz, Christian; Anz, David; Sandholzer, Nadja; Stecher, Bärbel; Rüssmann, Holger; Endres, Stefan; Bourquin, Carole

    2013-05-15

    The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4β7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4β7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.

  2. Loss of endothelial-ARNT in adult mice contributes to dampened circulating proangiogenic cells and delayed wound healing.

    Science.gov (United States)

    Han, Yu; Tao, Jiayi; Gomer, Alla; Ramirez-Bergeron, Diana L

    2014-12-01

    The recruitment and homing of circulating bone marrow-derived cells include endothelial progenitor cells (EPCs) that are critical to neovascularization and tissue regeneration of various vascular pathologies. We report here that conditional inactivation of hypoxia-inducible factor's (HIF) transcriptional activity in the endothelium of adult mice (Arnt(ΔiEC) mice) results in a disturbance of infiltrating cells, a hallmark of neoangiogenesis, during the early phases of wound healing. Cutaneous biopsy punches show distinct migration of CD31(+) cells into wounds of control mice by 36 hours. However, a significant decline in numbers of infiltrating cells with immature vascular markers, as well as decreased transcript levels of genes associated with their expression and recruitment, were identified in wounds of Arnt(ΔiEC) mice. Matrigel plug assays further confirmed neoangiogenic deficiencies alongside a reduction in numbers of proangiogenic progenitor cells from bone marrow and peripheral blood samples of recombinant vascular endothelial growth factor-treated Arnt(ΔiEC) mice. In addition to HIF's autocrine requirements in endothelial cells, our data implicate that extrinsic microenvironmental cues provided by endothelial HIF are pivotal for early migration of proangiogenic cells, including those involved in wound healing. © The Author(s) 2014.

  3. Sequential combined test, second trimester maternal serum markers, and circulating fetal cells to select women for invasive prenatal diagnosis.

    Directory of Open Access Journals (Sweden)

    Paolo Guanciali Franchi

    Full Text Available From January 1st 2013 to August 31st 2016, 24408 pregnant women received the first trimester Combined test and contingently offered second trimester maternal serum screening to identify those women who would most benefit from invasive prenatal diagnosis (IPD. The screening was based on first trimester cut-offs of ≥1:30 (IPD indicated, 1:31 to 1:899 (second trimester screening indicated and ≤1:900 (no further action, and a second trimester cut-off of ≥1:250. From January 2014, analysis of fetal cells from peripheral maternal blood was also offered to women with positive screening results. For fetal Down syndrome, the overall detection rate was 96.8% for a false-positive rate of 2.8% resulting in an odds of being affected given a positive result (OAPR of 1:11, equivalent to a positive predictive value (PPV of 8.1%. Additional chromosome abnormalities were also identified resulting in an OAPR for any chromosome abnormality of 1:6.6 (PPV 11.9%. For a sub-set of cases with positive contingent test results, FISH analysis of circulating fetal cells in maternal circulation identified 7 abnormal and 39 as normal cases with 100% specificity and 100% sensitivity. We conclude that contingent screening using conventional Combined and second trimester screening tests is effective but can potentially be considerably enhanced through the addition of fetal cell analysis.

  4. The Clinical Potential of Circulating Tumor Cells; The Need to Incorporate a Modern “Immunological Cocktail” in the Assay

    Energy Technology Data Exchange (ETDEWEB)

    Uhr, Jonathan W. [Cancer Immunobiology Center, University Texas Southwestern Medical Center, Dallas, TX 75390 (United States)

    2013-12-13

    The accepted clinical assay, CellSearch{sup ®}, and lab-on-a-chip tests for capturing circulating tumor cells are antibody-mediated. Attempts to improve their sensitivity have relied upon physical changes in the instruments. There have been no significant advances in improving the antibody-mediated portion of the capture. Modern immunologic engineering offers major possibilities for improving the sensitivity and other features of the assay. These include obtaining univalent antibody fragments such as scFvs with picomolar binding affinity and sufficient specificity; altering them to enhance their range of potential contact with target antigens; using antibodies directed against different epitopes on epithelial, mesenchymal or organ-specific cell surface markers to allow simultaneous binding and investigating non-antibody binding molecules as substitutes for antibody. These maneuvers could markedly improve the ability of current assays to improve patient care and might result in an acceptable test for detecting cancer earlier in high risk patients.

  5. The Clinical Potential of Circulating Tumor Cells; The Need to Incorporate a Modern “Immunological Cocktail” in the Assay

    Directory of Open Access Journals (Sweden)

    Jonathan W. Uhr

    2013-12-01

    Full Text Available The accepted clinical assay, CellSearch®, and lab-on-a-chip tests for capturing circulating tumor cells are antibody-mediated. Attempts to improve their sensitivity have relied upon physical changes in the instruments. There have been no significant advances in improving the antibody-mediated portion of the capture. Modern immunologic engineering offers major possibilities for improving the sensitivity and other features of the assay. These include obtaining univalent antibody fragments such as scFvs with picomolar binding affinity and sufficient specificity; altering them to enhance their range of potential contact with target antigens; using antibodies directed against different epitopes on epithelial, mesenchymal or organ-specific cell surface markers to allow simultaneous binding and investigating non-antibody binding molecules as substitutes for antibody. These maneuvers could markedly improve the ability of current assays to improve patient care and might result in an acceptable test for detecting cancer earlier in high risk patients.

  6. Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing.

    Science.gov (United States)

    Vaca-Paniagua, Felipe; Oliver, Javier; Nogueira da Costa, Andre; Merle, Philippe; McKay, James; Herceg, Zdenko; Holmila, Reetta

    2015-01-01

    To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

  7. Methods for continuous direct carbon fuel cell operation with a circulating electrolyte slurry

    Energy Technology Data Exchange (ETDEWEB)

    Harjes, Daniel I.; Dineen, Jr., D. Andrew; Guo, Liang; Calo, Joseph M.; Bloomfield, Valerie J.

    2017-02-07

    The present invention relates to methods and systems related to fuel cells, and in particular, to direct carbon fuel cells. The methods and systems relate to cleaning and removal of components utilized and produced during operation of the fuel cell, regeneration of components utilized during operation of the fuel cell, and generating power using the fuel cell.

  8. Fluorescence histochemistry of peptide hormone-producing cells: observations on the phenanthrenequinone method for the demonstration of arginine residues.

    Science.gov (United States)

    Sundler, F; Håkanson, R

    1978-07-12

    Phenanthrenequinone is a sensitive and specific fluorescence histochemical reagent for monosubstituted guanidines. It is probable that it selectively demonstrates the guanidino group of arginine residues of proteins and peptides. Phenanthrenequinone induces moderate to intense fluorescence in gastric chief cells, parenchymal cells of the pancreas, and certain peptide hormone-producing cell types such as the GH cells of the adenohypophysis and the glucagon cells of the pancreatic islets. Similar fluorescence spectra were obtained from an arginine-containing peptide in a histochemical model and from the GH cells of the adenohypophysis following exposure to phenanthrenequinone. We conclude that the cells demonstrated with this reagent store peptides or proteins rich in arginine.

  9. Circulating tumor cells as a biomarker of response to treatment in patient-derived xenograft mouse models of pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Robert J Torphy

    Full Text Available Circulating tumor cells (CTCs are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC, early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207 while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081. This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens.

  10. Tuberculosis in postchemotherapy residual masses in germ cell tumor of the testis

    Directory of Open Access Journals (Sweden)

    Rajesh Bansal

    2011-01-01

    Full Text Available Residual masses following chemotherapy in testicular tumors have been characterized as necrosis, mature or immature teratoma, and malignant tumors. Twenty four patients had retroperitoneal lymph node dissection for postchemotherapy residual masses between January 2000 and December 2008. We report two patients; one with late relapse and other with postchemotherapy residual mass, who had tuberculosis. Tumor markers were normal, and PET scan showed increased uptake in residual mass. There are no previous reports of tuberculosis in postchemotherapy residual masses.

  11. Etiopathogenesis of type 1 diabetes mellitus: prognostic factors for the evolution of residual β cell function

    Directory of Open Access Journals (Sweden)

    Dib Sergio A

    2009-12-01

    Full Text Available Abstract Type 1A diabetes mellitus (T1ADM is a progressive autoimmune disease mediated by T lymphocytes with destruction of beta cells. Up to now, we do not have precise methods to assess the beta cell mass, "in vivo" or "ex-vivo". The studies about its genetic susceptibility show strong association with class II antigens of the HLA system (particularly DQ. Others genetics associations are weaker and depend on the population studied. A combination of precipitating events may occur at the beginning of the disease. There is a silent loss of immune-mediated beta cells mass which velocity has an inverse relation with the age, but it is influenced by genetic and metabolic factors. We can predict the development of the disease primarily through the determination of four biochemically islet auto antibodies against antigens like insulin, GAD65, IA2 and Znt8. Beta cell destruction is chronically progressive but at clinical diagnosis of the disease a reserve of these cells still functioning. The goal of secondary disease prevention is halt the autoimmune attack on beta cells by redirecting or dampening the immune system. It is remains one of the foremost therapeutic goals in the T1ADM. Glycemic intensive control and immunotherapeutic agents may preserve beta-cell function in newly diagnosed patients with T1ADM. It may be assessed through C-peptide values, which are important for glycemic stability and for the prevention of chronic complications of this disease. This article will summarize the etiopathogenesis mechanisms of this disease and the factors can influence on residual C-peptide and the strategies to it preservation.

  12. Circulating CXCR5⁺CD4⁺ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines.

    Science.gov (United States)

    Matsui, Ken; Adelsberger, Joseph W; Kemp, Troy J; Baseler, Michael W; Ledgerwood, Julie E; Pinto, Ligia A

    2015-01-01

    Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like) cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as

  13. Distribution of residual cancer cells in the bowel wall after neoadjuvant chemoradiation in patients with rectal cancer.

    Science.gov (United States)

    Duldulao, Marjun P; Lee, Wendy; Streja, Leanne; Chu, Peiguo; Li, Wenyan; Chen, Zhenbin; Kim, Joseph; Garcia-Aguilar, Julio

    2013-02-01

    The standard treatment for locally advanced rectal cancer is preoperative chemoradiation and total mesorectal excision. After surgery, tumors are classified according to the depth of tumor invasion, nodal involvement, and tumor regression grade. However, these staging systems do not provide information about the distribution of residual cancer cells within the bowel wall. This study aimed to determine the distribution of residual cancer cells in each layer of the bowel wall in rectal cancer specimens. This was a secondary analysis of data from a prospective phase II study. This study was performed in a multi-institutional setting. Included were 153 patients with stage II or stage III rectal cancer. Patients were treated with chemoradiation and surgery. The surgical specimen tumor tissue was analyzed, and the distribution of residual cancer cells in each layer of the bowel wall was determined. Statistical analysis was used to examine the correlation of residual cancer cells in each layer of the bowel wall with the clinical/pathologic stage and tumor regression grade. Forty-two of 153 (27%) patients had complete response in the bowel wall (ypT0). Of the remaining 111 patients who had residual cancer cells, 5 (3%) were ypTis, 12 (8%) were ypT1, 41 (27%) were ypT2, 50 (33%) were ypT3, and 3 (2%) were ypT4. Of the 94 patients with ypT2-4 tumors, 12 (13%) had cancer cells in the mucosa, and 53 (56%) had cancer cells in the submucosa; 92 (98%) had cancer cells in the muscularis propria. Pretreatment cT correlated with the distribution of residual cancer cells. Tumor regression grade was not associated with the distribution of residual cancer cells after chemoradiation. : Patients received different chemotherapy regimens. Residual cancer cells in rectal cancer specimens after chemoradiation are preferentially located close to the invasive front. This should be considered when designing strategies to diagnose complete pathologic response and when investigating the

  14. Circulating intercellular adhesion molecule-1 (ICAM-1) as an early and sensitive marker for virus-induced T cell activation

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Johansen, J; Marker, O

    1995-01-01

    mice, clearly demonstrating that T cells were mandatory. Analysis of MHC class I and MHC class II-deficient mice revealed that either CD4+ or CD8+ T cells alone are sufficient, despite a markedly reduced inflammatory exudate in the former animals. These results indicate that virus-activated T cells......The effect of systemic virus infection on the level of circulating ICAM-1 (cICAM-1) in serum, and the role of virus-activated T cells in this context, were studied using the murine lymphocytic choriomeningitis virus infection as primary model system. A marked virus-induced elevation in cICAM-1...... in serum was revealed, the presence of which coincided with the phase of virus-induced T cell activation. However, high levels of cICAM-1 in serum were observed well before maximal T cell activation could be demonstrated. No increase in cICAM-1 was observed in the serum of infected T cell-deficient nude...

  15. Detection of Lipid-Rich Prostate Circulating Tumour Cells with Coherent Anti-Stokes Raman Scattering Microscopy

    International Nuclear Information System (INIS)

    Mitra, Ranjana; Chao, Olivia; Urasaki, Yasuyo; Goodman, Oscar B; Le, Thuc T

    2012-01-01

    Circulating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge. Coherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line. One hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells. Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC

  16. Usefulness of transcription-reverse transcription concerted reaction method for detecting circulating tumor cells in patients with colorectal cancer.

    Science.gov (United States)

    Sato, Nobutaka; Hayashi, Naoko; Imamura, Yu; Tanaka, Yohei; Kinoshita, Koichi; Kurashige, Jyunji; Saito, Seiya; Karashima, Ryuichi; Hirashima, Kotaro; Nagai, Yohei; Miyamoto, Yuji; Iwatsuki, Masaaki; Baba, Yoshifumi; Watanabe, Masayuki; Baba, Hideo

    2012-06-01

    The CellSearch system (Veridex, LLC) is useful for detecting circulating tumor cells (CTCs) in various carcinomas, including colorectal cancer (CRC); however, there are some problems associated with its clinical use. A transcription-reverse transcription concerted reaction (TRC) method, which is a PCR-based technique producing more stable and reliable results, because it is a more simplified process compared with the conventional techniques, has been introduced for detecting micrometastasis in some carcinomas. We aimed to demonstrate the effectiveness of TRC method in the CTC detection. We compared the two methods for the sensitivity for CTC detection using the colon cancer cell line and 42 whole-blood samples from patients with advanced or metastatic CRC. Furthermore, 25 patients with metastatic CRC were enrolled to investigate the correlation between CTC detection and prognosis in both methods. The sensitivity of the TRC method was similar to that of the CellSearch system. The overall survival rate was significantly worse in the patients diagnosed as CTC-positive by the TRC method than in those diagnosed as CTC-negative; this finding was similar to the prognosis indicated by the CellSearch system. However, clinically, the TRC method could detect CTCs more rapidly and at a reduced cost compared with the CellSearch system. The TRC method seems to be a useful alternative to the CellSearch system for clinically detecting CTCs in patients with metastatic CRC.

  17. A Randomized, Controlled Pilot Study of the Effects of Acupuncture on Circulating Endothelial Progenitor Cells in Coronary Heart Disease.

    Science.gov (United States)

    Painovich, Jeannette; Phancao, Anita; Mehta, Puja; Chowdhury, Supurna; Dhawan, Shivani; Li, Ning; Taylor, Doris; Qiao, Yi; Brantman, Anna; Ma, Xiuling; Merz, C Noel Bairey

    2014-04-01

    Coronary heart disease (CHD) remains the number one killer of men and women in the United States, and despite traditional secondary prevention, individuals with the disease remain at risk. Endothelial progenitor cells (EPCs) may have beneficial effects on atherosclerosis, angiogenesis, and vascular repair and may contribute systemically to ongoing endogenous repair processes. Traditional acupuncture (TA), a modality used in the practice of Chinese medicine, appears to have beneficial effects in many areas associated with CHD. The study examined the effects of TA on circulating EPCs in individuals with CHD. The research team performed a randomized, controlled pilot study. All interventions were performed at the Cedars-Sinai Medical Center in Los Angeles, CA. The study included 13 participants in 3 groups: (1) TA (n = 5), (2) sham acupuncture (SA, n = 5), or (3) waiting control (WC, n = 3). The TA group received acupuncture treatments for 12 wk at CHD-specific sites, while the SA group received no-needle pressure at nonacupuncture sites for the same period, and the WC group received no intervention. The study measured the number of EPCs circulating in peripheral blood to determine cell surface markers for expressions of cluster of differentiation 34, 133 (CD34+/CD133+) and vascular endothelial growth factor receptor 2 (VEGF-R2+). Eight men and 5 women with a mean age of 59 ± 10.9 y were included. Compared with their measurements at baseline, members of the TA group had a significantly greater change in the level of EPCs expressing CD34+/VEGF-R2+ compared with the SA group (P = .04). No group differences were evident in immature EPCs expressing CD34+/CD133+. The study's results suggest that TA can alter the number of EPCs circulating in peripheral blood by increasing the mobilization of the VEGF-R2+ EPC subpopulations. Further studies are warranted to evaluate whether TA can beneficially affect CHD via augmentation of EPC regenerative pathways.

  18. Prognostic significance of circulating tumor cells in esophageal carcinoma: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Qiao GL

    2016-03-01

    Full Text Available Guang-Lei Qiao,1 Wei-Xiang Qi,2 Wei-Hua Jiang,1 Ying Chen,1 Li-Jun Ma1 1Department of Oncology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, 2Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Shanghai, People’s Republic of China Purpose: The prognostic significance of circulating tumor cells (CTCs in esophageal carcinoma (EC is controversial. We aim to assess its association with clinicopathological and prognostic relevance in EC by using a meta-analysis.Methods: We searched PubMed, Cochrane Database, Embase databases, and the references in relevant studies that assessed the clinicopathological or prognostic relevance of CTCs in peripheral blood of patients with EC. Statistical analyses were conducted by using Stata software to calculate the pooled odds ratio (OR, hazard ratio (HR, and 95% confidence intervals (CIs using fixed or random-effects models according to the heterogeneity of included studies. The subgroup analyses were performed according to ethnicity, histological type, and detection method.Results: Sixteen trials containing 1,260 patients were included for analysis. Pooled results showed that presence of CTCs was significantly associated with poor overall survival (HR =1.71, 95% CI [1.30, 2.12], P<0.001 and progression-free survival (HR =1.67, 95% CI [1.19, 2.15], P<0.001 in EC patients. Subgroup analysis indicated that presence of CTCs was closely associated with worse overall survival (Asian: HR =1.66, 95% CI [1.24, 2.08], P<0.001; squamous cell carcinoma [SCC]: HR =1.66, 95% CI [1.24, 2.08], P<0.001; no polymerase chain reaction [PCR]: HR =2.08, 95% CI [1.40, 2.76], P<0.001 and progression-free survival (Asian: HR =1.63, 95% CI [1.15, 2.12], P<0.001; SCC: HR =1.63, 95% CI [1.15, 2.12], P<0.001; PCR: HR =1.63, 95% CI [1.15, 2.12], P<0.001. Additionally, ORs showed that presence of CTCs was significantly correlated with tumor node metastasis (TNM staging (overall: OR =1

  19. Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection.

    Directory of Open Access Journals (Sweden)

    Jia-Yang Chen

    Full Text Available Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB "smart coating" to capture viable circulating tumor cells (CTCs and circulating tumor microemboli (CTM directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

  20. Profiling the effects of process changes on residual host cell proteins in biotherapeutics by mass spectrometry.

    Science.gov (United States)

    Schenauer, Matthew R; Flynn, Gregory C; Goetze, Andrew M

    2013-01-01

    An advanced liquid chromatography/mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance (DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP composition of two upstream samples was also analyzed and used to demonstrate that HCPs that carry through purification processes to be detectable in DS are not always among those that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating process development as well as more rationally assessing potential safety risks posed by individual, identified HCPs. © 2013 American Institute of Chemical Engineers.

  1. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    Science.gov (United States)

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  2. Circulating CD3+CD4+CD161+ Cells Are Associated with Early Complications after Autologous Stem Cell Transplantation in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Sung-Eun Lee

    2018-01-01

    Full Text Available The aim of this study was to explore if measurement of pretransplant circulating CD161-expressing cells, in addition to clinical risk factors, could predict mucositis and infections in patients with multiple myeloma (MM undergoing autologous stem cell transplantation (ASCT. To determine if CD161-expressing cells are likely to predict early complications, namely, mucositis (≥grade 3, infections, and cytomegalovirus (CMV reactivation, we prospectively examined CD161-expressing cells (CD3+CD4+CD161+ and CD3+CD8+CD161+ in peripheral blood samples from 108 patients with MM undergoing ASCT. After adjusting for factors identified by univariate analysis that predicted mucositis (≥grade 3, infection before engraftment, and CMV reactivation, multivariate analyses revealed that the low proportion of CD3+CD4+CD161+ cells in peripheral blood was an independent predictor of mucositis (≥grade 3 (P=0.020, infections before engraftment (P=0.014, and CMV reactivation (P=0.010. In addition, we found that female sex and decreased glomerular filtration rate were independent factors for predicting mucositis. Female sex and severe pulmonary comorbidity were independent factors for predicting infection before engraftment. We found that the proportion of circulating CD3+CD4+CD161+ cells is useful for predicting the occurrence of early complications, including mucositis and infections, after ASCT in patients with MM.

  3. Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection

    OpenAIRE

    Shi, Jian; Luo, Fengbao; Shi, Qianqian; Xu, Xianlin; He, Xiaozhou; Xia, Ying

    2015-01-01

    Background Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study. Methods The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules inc...

  4. Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Roodselaar, Jay; Evans, Matthew C; Gardiner, Chris; Sargent, Ian; Romero, Ignacio A; Bristow, Adrian; Buchan, Alastair M; Haughey, Norman; Anthony, Daniel C

    2017-08-29

    Brain injury elicits a systemic acute-phase response (APR), which is responsible for co-ordinating the peripheral immunological response to injury. To date, the mechanisms responsible for signalling the presence of injury or disease to selectively activate responses in distant organs were unclear. Circulating endogenous extracellular vesicles (EVs) are increased after brain injury and have the potential to carry targeted injury signals around the body. Here, we examined the potential of EVs, isolated from rats after focal inflammatory brain lesions using IL-1β, to activate a systemic APR in recipient naïve rats, as well as the behavioural consequences of EV transfer. Focal brain lesions increased EV release, and, following isolation and transfer, the EVs were sequestered by the liver where they initiated an APR. Transfer of blood-borne EVs from brain-injured animals was also enough to suppress exploratory behaviours in recipient naïve animals. EVs derived from brain endothelial cell cultures treated with IL-1β also activated an APR and altered behaviour in recipient animals. These experiments reveal that inflammation-induced circulating EVs derived from endothelial cells are able to initiate the APR to brain injury and are sufficient to generate the associated sickness behaviours, and are the first demonstration that EVs are capable of modifying behavioural responses.

  5. Microfluidic flow fractionation device for label-free isolation of circulating tumor cells (CTCs) from breast cancer patients.

    Science.gov (United States)

    Hyun, Kyung-A; Kwon, Kiho; Han, Hyunju; Kim, Seung-Il; Jung, Hyo-Il

    2013-02-15

    Circulating tumor cells (CTCs) are dissociated from primary tumor and circulate in peripheral blood. They are regarded as the genesis of metastasis. Isolation and enumeration of CTCs serve as valuable tools for cancer prognosis and diagnosis. However, the rarity and heterogeneity of CTCs in blood makes it difficult to separate intact CTCs without loss. In this paper, we introduce a parallel multi-orifice flow fractionation (p-MOFF) device in which a series of contraction/expansion microchannels are placed parallel on a chip forming four identical channels. CTCs were continuously isolated from the whole blood of breast cancer patients by hydrodynamic forces and cell size differences. Blood samples from 24 breast cancer patients were analyzed (half were from metastatic breast cancer patients and the rest were from adjuvant breast cancer patients). The number of isolated CTCs varied from 0 to 21 in 7.5 ml of blood. Because our devices do not require any labeling processes (e.g., EpCAM antibody), heterogeneous CTCs can be isolated regardless of EpCAM expression. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Circulating Differentially Methylated Amylin DNA as a Biomarker of β-Cell Loss in Type 1 Diabetes.

    Directory of Open Access Journals (Sweden)

    John A Olsen

    Full Text Available In type 1 diabetes (T1D, β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss.

  7. Glycan Stimulation Enables Purification of Prostate Cancer Circulating Tumor Cells on PEDOT NanoVelcro Chips for RNA Biomarker Detection.

    Science.gov (United States)

    Shen, Mo-Yuan; Chen, Jie-Fu; Luo, Chun-Hao; Lee, Sangjun; Li, Cheng-Hsuan; Yang, Yung-Ling; Tsai, Yu-Han; Ho, Bo-Cheng; Bao, Li-Rong; Lee, Tien-Jung; Jan, Yu Jen; Zhu, Ya-Zhen; Cheng, Shirley; Feng, Felix Y; Chen, Peilin; Hou, Shuang; Agopian, Vatche; Hsiao, Yu-Sheng; Tseng, Hsian-Rong; Posadas, Edwin M; Yu, Hsiao-Hua

    2018-02-01

    A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival

    Directory of Open Access Journals (Sweden)

    Kuhlmann Jan

    2012-07-01

    Full Text Available Abstract Background We recently showed that LOH proximal to M6P/IGF2R locus (D6S1581 in primary ovarian tumors is predictive for the presence of disseminated tumor cells (DTC in the bone marrow (BM. For therapy-monitoring, it would be highly desirable to establish a blood-based biomarker. Therefore, we quantified circulating DNA (cirDNA in sera of 63 ovarian cancer patients before surgery and after chemotherapy, measured incidence of LOH at four cancer-relevant chromosomal loci, correlated LOH with tumor cell spread to the BM and evaluated prognostic significance of LOH. Methods cirDNA was fractionated into high- and low molecular-weight fraction (HMWF, LMWF for LOH-profiling, utilizing PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results cirDNA levels in the HMWF before surgery were predictive for residual tumor load (p = 0.017. After chemotherapy, we observed a significant decline of cirDNA in the LMWF (p = 0.0001 but not in the HMWF. LOH was prevalently detected in the LMWF with an overall frequency of 67%, only moderately ablating after chemotherapy (45%. Before surgery, LOH in the LMWF at marker D10S1765 and D13S218 significantly correlated with tumor grading and FIGO stage (p = 0.033, p = 0.004, respectively. In both combined fractions, LOH at D6S1581 additionally associated with overall survival (OS (p = 0.030. Moreover, solely LOH at D10S1765 in LMWF after therapy correlated with DTC in BM after therapy (p = 0.017. Conclusion We demonstrate the applicability and necessity of DNA-fractionation prior to analyzing circulating LOH and identify LOH at D10S1765 and D6S1581 as novel blood-based biomarkers for ovarian cancer, being relevant for therapy-monitoring.

  9. Residual ß-cell function and microvascular complications in type 1 diabetic patients

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    Gomes M.B.

    2000-01-01

    Full Text Available To determine the influence of residual ß-cell function on retinopathy and microalbuminuria we measured basal C-peptide in 50 type 1 diabetic outpatients aged 24.96 ± 7.14 years, with a duration of diabetes of 9.1 ± 6.2 years. Forty-three patients (86% with low C-peptide (<0.74 ng/ml had longer duration of diabetes than 7 patients (14% with high C-peptide (³0.74 ng/ml (9 (2-34 vs 3 (1-10 years, P = 0.01 and a tendency to high glycated hemoglobin (HBA1 (8.8 (6-17.9 vs 7.7 (6.9-8.7%, P = 0.08. Nine patients (18% had microalbuminuria (two out of three overnight urine samples with an albumin excretion rate (AER ³20 and <200 µg/min and 13 (26% had background retinopathy. No association was found between low C-peptide, microalbuminuria and retinopathy and no difference in basal C-peptide was observed between microalbuminuric and normoalbuminuric patients (0.4 ± 0.5 vs 0.19 ± 0.22 ng/ml, P = 0.61 and between patients with or without retinopathy (0.4 ± 0.6 vs 0.2 ± 0.3 ng/ml, P = 0.43. Multiple regression analysis showed that duration of diabetes (r = 0.30, r2 = 0.09, P = 0.031 followed by HBA1 (r = 0.41, r2 = 0.17, P = 0.01 influenced basal C-peptide, and this duration of diabetes was the only variable affecting AER (r = 0.40, r2 = 0.16, P = 0.004. In our sample of type 1 diabetic patients residual ß-cell function was not associated with microalbuminuria or retinopathy.

  10. Positive HER-2 protein expression in circulating prostate cells and micro-metastasis, resistant to androgen blockage but not diethylstilbestrol

    Directory of Open Access Journals (Sweden)

    Nigel P Murray

    2011-01-01

    Full Text Available Introduction : HER-2 expression in prostate cancer is associated with a worse prognosis and is suggested to play a role in androgen resistance. We present a study of HER-2 expression in circulating tumor cells and micrometastasis in bone marrow and the effect of androgen blockage or DES in the presence of HER-2 expressing cells. Patients and Methods : A multicenter study of men with prostate cancer, treated with surgery, radiotherapy, or observation, and with or without hormone therapy. Mononuclear cells were separated from blood and bone marrow aspirate by differential centrifugation, touch preps were made from bone marrow biopsy samples. Prostate cells were detected using anti-PSA monoclonal antibody and standard immunocytochemistry. Positive samples were processed using Herceptest® to determine HER-2 expression. After 1 year, patients were re-evaluated and the findings of HER-2 expression and PSA change compared with treatment. Results : Total 199 men participated, and 97 had a second evaluation 1 year later, frequency of HER-2 expression in circulating tumor cells and micrometastasis was 18% and 21%, respectively. There was no significant difference in HER-2 expression in the pretreatment group, after radical surgery or radiotherapy or with biochemical failure. Men with androgen blockade had a significantly higher expression of HER-2 (58% (P =0.001. Of the 97 men with a second evaluation, 56 were in the observation arm, 27 androgen blockade, and 14 DES. Use of androgen blockade or DES significantly reduced serum PSA levels in comparison with observation (P =0.001. However, there was a significant increase in HER-2 expression in patients with androgen blockade (P =0.05 en comparison with observation or DES treatment. No patient with observation or DES became HER-2 positive, en comparison 4/22 patients initially HER-2 negative became HER-2 positive with androgen blockade. Conclusions : The results suggest that HER-2 positive cells are

  11. A new marker set that identifies fetal cells in maternal circulation with high specificity

    DEFF Research Database (Denmark)

    Hatt, Lotte; Brinch, Marie; Singh, Ripudaman

    2014-01-01

    were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. RESULTS: Fetal...... cell candidates could be detected in 91% of the samples, and in 85% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100% if three...

  12. Circulating Regulatory T-Cells in Monoclonal Gammopathies of Uncertain Significance and Multiple Myeloma: In Search of a Role

    Directory of Open Access Journals (Sweden)

    Giovanni D’Arena

    2016-01-01

    Full Text Available The frequency and function of regulatory T-cells (Tregs in multiple myeloma (MM are still matter of debate. The percentage and absolute number of circulating Tregs (CD4+CD25+high  densityCD127-/low  density from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1%  ± 1.0 (range 0.75–6.1% in MM patients; 2.1%  ± 0.9 (range 0.3–4.4% in MGUS; and 1.5%  ± 0.4 (range 0.9–2.1% in controls (p ns. Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7–149/μL in MM; 38.8/μL ± 19.1 (range 4.3–87/μL in MGUS; and 39.4/μL ± 12.5 (range 18–63/μL in controls (p ns. After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM.

  13. Prior endurance exercise prevents postprandial lipaemia-induced increases in reactive oxygen species in circulating CD31+ cells.

    Science.gov (United States)

    Jenkins, Nathan T; Landers, Rian Q; Thakkar, Sunny R; Fan, Xiaoxuan; Brown, Michael D; Prior, Steven J; Spangenburg, Espen E; Hagberg, James M

    2011-11-15

    We hypothesized that prior exercise would prevent postprandial lipaemia (PPL)-induced increases in intracellular reactive oxygen species (ROS) in three distinct circulating angiogenic cell (CAC) subpopulations. CD34(+), CD31(+)/CD14(-)/CD34(-), and CD31(+)/CD14(+)/CD34(-) CACs were isolated from blood samples obtained from 10 healthy men before and 4 h after ingesting a high fat meal with or without ∼50 min of prior endurance exercise. Significant PPL-induced increases in ROS production in both sets of CD31(+) cells were abolished by prior exercise. Experimental ex vivo inhibition of NADPH oxidase activity and mitochondrial ROS production indicated that mitochondria were the primary source of PPL-induced oxidative stress. The attenuated increases in ROS with prior exercise were associated with increased antioxidant gene expression in CD31(+)/CD14(-)/CD34(-) cells and reduced intracellular lipid uptake in CD31(+)/CD14(+)/CD34(-) cells. These findings were associated with systemic cardiovascular benefits of exercise, as serum triglyceride, oxidized low density lipoprotein-cholesterol, and plasma endothelial microparticle concentrations were lower in the prior exercise trial than the control trial. In conclusion, prior exercise completely prevents PPL-induced increases in ROS in CD31(+)/CD14(-)/CD34(-) and CD31(+)/CD14(+)/CD34(-) cells. The mechanisms underlying the effects of exercise on CAC function appear to vary among specific CAC types.

  14. Dual-patterned immunofiltration (DIF) device for the rapid efficient negative selection of heterogeneous circulating tumor cells.

    Science.gov (United States)

    Bu, Jiyoon; Kang, Yoon-Tae; Kim, Young Jun; Cho, Young-Ho; Chang, Hee Jin; Kim, Hojoong; Moon, Byung-In; Kim, Ho Gak

    2016-11-29

    The analysis of circulating tumor cells (CTCs) is an emerging field for estimating the metastatic relapse and tumor burden of cancer patients. However, the isolation of CTCs is still challenging due to their ambiguity, rarity, and heterogeneity. Here, we present an anti-CD45 antibody based dual-patterned immunofiltration (DIF) device for the enrichment of heterogeneous CTC subtypes by effective elimination of leukocytes. Our uniquely designed dual-patterned layers significantly enhance the binding chance between immuno-patterns and leukocytes due to the fluidic whirling and the increased binding sites, thus achieving superior negative selection in terms of high-throughput and high purity. From the experiments using lung cancer cells, 97.07 ± 2.79% of leukocytes were eliminated with less than 10% loss of cancerous cells at the flow rate of 1 mL h -1 . To verify the device as a potential diagnostic tool, CTCs were collected from 11 cancer patients' blood and an average of 283.3 CTC-like cells were identified while less than 1 CTC-like cells were found from healthy donors. The samples were also analyzed by immunohistochemistry and the reverse transcription polymerase chain reaction to identify their heterogeneous characteristics. These remarkable results demonstrate that the present device could help to understand the unknown properties or undiscovered roles of CTCs with a non-biased view.

  15. Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

    Science.gov (United States)

    Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

    2016-04-01

    Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

  16. Multivariate analysis of T-cell functional defects and circulating serum factors in Hodgkin's disease.

    Science.gov (United States)

    Schulof, R S; Bockman, R S; Garofalo, J A; Cirrincione, C; Cunningham-Rundles, S; Fernandes, G; Day, N K; Pinsky, C M; Incefy, G S; Thaler, H T; Good, R A; Gupta, S

    1981-08-15

    A comprehensive immunologic and serologic analysis was performed on 31 untreated patients with Hodgkin's disease. Immune evaluations stressed T-cell functional activity and included traditional parameters (PHA responsiveness and delayed hypersensitivity skin reactivity), as well as newer functional assays (T-cell colony formation, chemotaxis, spontaneous and antibody-dependent cytotoxicity, and concanavalin A-induced suppressor cell activity (CISA). Serum factors included ferritin, prostaglandins, zinc, copper, immune complexes, and thymic hormone activity. Every patient exhibited at least one T-cell or serum abnormality. The greatest percentage of patients exhibited T-cell defects in chemotaxis (85%), colony formation (81%). and PHA reactivity (64%). Immune defects were more common with advanced disease but were not related to absolute T-cell or monocyte count, skin test anergy, or abnormalities of T mu/T gamma cell proportions. Linear relationships were identified among abnormalities in the three assays employing mononuclear cells (PHA, colony formation, CISA) which may have reflected the inhibitory influence of monocytes present in the mononuclear cell preparations. Low serum zinc correlated with marked impairment of T-cell chemotaxis. Elevated prostaglandins were associated with high PHA reactivity and with depressed colony formation. Our results indicate that many complex factors, including intrinsic T-cell defects, contribute to the impaired immunity associated with Hodgkin's disease.

  17. Circulating lipocalin 2 is neither related to liver steatosis in patients with non-alcoholic fatty liver disease nor to residual liver function in cirrhosis.

    Science.gov (United States)

    Meier, Elisabeth M; Pohl, Rebekka; Rein-Fischboeck, Lisa; Schacherer, Doris; Eisinger, Kristina; Wiest, Reiner; Krautbauer, Sabrina; Buechler, Christa

    2016-09-01

    Lipocalin 2 (LCN2) is induced in the injured liver and associated with inflammation. Aim of the present study was to evaluate whether serum LCN2 is a non-invasive marker to assess hepatic steatosis in patients with non-alcoholic fatty liver disease (NAFLD) or residual liver function in patients with liver cirrhosis. Therefore, LCN2 was measured by ELISA in serum of 32 randomly selected patients without fatty liver (controls), 24 patients with ultrasound diagnosed NAFLD and 42 patients with liver cirrhosis mainly due to alcohol. Systemic LCN2 was comparable in patients with liver steatosis, those with liver cirrhosis and controls. LCN2 negatively correlated with bilirubin in both cohorts. In cirrhosis, LCN2 was not associated with more advanced liver injury defined by the CHILD-PUGH score and model for end-stage liver disease score. Resistin but not C-reactive protein or chemerin positively correlated with LCN2. LCN2 levels were not increased in patients with ascites or patients with esophageal varices. Consequently, reduction of portal pressure by transjugular intrahepatic portosystemic shunt did not affect LCN2 levels. Hepatic venous blood (HVS), portal venous blood and systemic venous blood levels of LCN2 were similar. HVS LCN2 was unchanged in patients with end-stage liver cirrhosis compared to those with well-compensated disease arguing against increased hepatic release. Current data exclude that serum LCN2 is of any value as steatosis marker in patients with NAFLD and indicator of liver function in patients with alcoholic liver cirrhosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Bioengineered fibrin-based niche to direct outgrowth of circulating progenitors into neuron-like cells for potential use in cellular therapy

    Science.gov (United States)

    Tara, S.; Krishnan, Lissy K.

    2015-06-01

    Objective. Autologous cells are considered to be the best choice for use in transplantation therapy. However, the challenges and risks associated with the harvest of transplantable autologous cells limit their successful therapeutic application. The current study explores the possibility of isolating neural progenitor cells from circulating multipotent adult progenitor cells for potential use in cell-based and patient-specific therapy for neurological diseases. Approach. To enable the selection of neural progenitor cells from human peripheral blood mononuclear cells, and to support their lineage maintenance, the composition of a fibrin-based niche was optimized. Morphological examination and specific marker analysis were carried out, employing a qualitative/quantitative polymerase chain reaction followed by immunocytochemistry to: (i) characterize neural progenitor cells in culture; (ii) monitor proliferation/survival; and (iii) track their differentiation status. Main results. The presence of neural progenitors in circulation was confirmed by the presence of nestin+ cells at the commencement of the culture. The isolation, proliferation and differentiation of circulating neural progenitors to neuron-like cells were directed by the engineered niche. Neural cell isolation to near homogeneity was confirmed by the expression of β-III tubulin in ∼95% of cells, whereas microtubule associated protein-2 expression confirmed their ability to differentiate. The concentration of potassium chloride in the niche was found to favour neuron-like cell lengthening, cell-cell contact, and expressions of synaptophysin and tyrosine hydroxylase. Significance. The purpose of this research was to find out if peripheral blood could serve as a potential source of neural progenitors for cell based therapy. The study established that neural progenitors could be selectively isolated from peripheral blood mononuclear cells using a biomimetic niche. The selected cells could multiply and

  19. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1.

    Directory of Open Access Journals (Sweden)

    Jennifer L Schehr

    Full Text Available Expression of programmed-death ligand 1 (PD-L1 in non-small cell lung cancer (NSCLC is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT.To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP technology.Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33-100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples.Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the

  20. Diagnostic yield of primary circulating tumor cells in women suspected of breast cancer: the BEST (Breast Early Screening Test) study.

    Science.gov (United States)

    Murray, Nigel P; Miranda, Roxana; Ruiz, Amparo; Droguett, Elsa

    2015-01-01

    To determine the diagnostic yield of primary circulating tumor cells in women with suspicion of breast cancer, detected as a result of an abnormal mammography. Consecutive women presenting for breast biopsy as a result of a mammogram BiRADs of 3 or more, had an 8 ml blood sample taken for primary circulating tumor cell (CTC) detection. Mononuclear cells were obtained using differential gel centrifugation and CTCs identified using standard immunocytochemistry using anti-mammoglobin. A test was determined to be positive if 1 CTC was detected. A total of 144 women with a mean age of 54.7 ± 15.6 years participated, 78/144 (53.0%) had breast cancer on biopsy, 65/140 (46.3%) benign pathologies and 1(0.7%) non-Hogkins lymphoma. Increasing BiRADs scores were associated with increased cancer detection (p=0.004, RR 1.00, 4.24, 8.50). CTC mammoglobin positive had a sensitivity of 81.1% and specificity of 90.9%, with positive and negative predictive values of 90.9% and 81.1% respectively. Mammoglobin positive CTCs detected 87% of invasive cancers, while poorly differentiated cancers were negative for mammoglobin. Only 50% of in situ cancers and none of the intraductal cancers had CTCs detected. Menopausal status did not affect the diagnostic yield of the CTC test, which was higher in women with BiRADS 4 mammograms. There was a significant trend (pBIRADs 4a mammagrams and in the detection of interval invasive breast cancer, larger studies are needed.

  1. Inflammation-Induced Changes in Circulating T-Cell Subsets and Cytokine Production During Human Endotoxemia

    DEFF Research Database (Denmark)

    Ronit, Andreas; Plovsing, Ronni R; Gaardbo, Julie C

    2017-01-01

    Observational clinical studies suggest the initial phase of sepsis may involve impaired cellular immunity. In the present study, we investigated temporal changes in T-cell subsets and T-cell cytokine production during human endotoxemia. Endotoxin (Escherichia coli lipopolysaccharide 4 ng....../kg) was administered intravenously in 15 healthy volunteers. Peripheral blood and bronchoalveolar lavage fluid (BALF) were collected at baseline and after 2, 4, 6, 8, and 24 hours for flow cytometry. CD4(+)CD25(+)CD127lowFoxp3(+) regulatory T cells (Tregs), CD4(+)CD161(+) cells, and activated Human leukocyte antigen......, HLA-DR(+)CD38(+) T cells were determined. Ex vivo whole-blood cytokine production and Toll-like receptor (TLR)-4 expression on Tregs were measured. Absolute number of CD3(+)CD4(+) (P = .026), CD3(+)CD8(+) (P = .046), Tregs (P = .023), and CD4(+)CD161(+) cells (P = .042) decreased after endotoxin...

  2. Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding.

    Science.gov (United States)

    Elsheikh, E; Andersson, E; Sylvén, C; Ericzon, B-G; Palmblad, J; Mints, M

    2014-01-01

    Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P Market Insurance. The authors have no conflict of interest to declare.

  3. Prognostic Impact of Modulators of G proteins in Circulating Tumor Cells from Patients with Metastatic Colorectal Cancer.

    Science.gov (United States)

    Barbazan, Jorge; Dunkel, Ying; Li, Hongying; Nitsche, Ulrich; Janssen, Klaus-Peter; Messer, Karen; Ghosh, Pradipta

    2016-02-26

    The consequence of a loss of balance between G-protein activation and deactivation in cancers has been interrogated by studying infrequently occurring mutants of trimeric G-protein α-subunits and GPCRs. Prior studies on members of a newly identified family of non-receptor guanine nucleotide exchange factors (GEFs), GIV/Girdin, Daple, NUCB1 and NUCB2 have revealed that GPCR-independent hyperactivation of trimeric G proteins can fuel metastatic progression in a variety of cancers. Here we report that elevated expression of each GEF in circulating tumor cells (CTCs) isolated from the peripheral circulation of patients with metastatic colorectal cancer is associated with a shorter progression-free survival (PFS). The GEFs were stronger prognostic markers than two other markers of cancer progression, S100A4 and MACC1, and clustering of all GEFs together improved the prognostic accuracy of the individual family members; PFS was significantly lower in the high-GEFs versus the low-GEFs groups [H.R = 5, 20 (95% CI; 2,15-12,57)]. Because nucleotide exchange is the rate-limiting step in cyclical activation of G-proteins, the poor prognosis conferred by these GEFs in CTCs implies that hyperactivation of G-protein signaling by these GEFs is an important event during metastatic progression, and may be more frequently encountered than mutations in G-proteins and/or GPCRs.

  4. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  5. Stimulation of murine stem cell proliferation by circulating activities produced during the recovery of a radiation-induced hemopoietic injury

    International Nuclear Information System (INIS)

    Grande Azanedo, M.T.

    1988-01-01

    The proliferative activity of CFU-S, low in normal steady state, increases after treatment with different aggressors, i.e. radiation. This stimulation has been attributed in part to a local regulation system of stem cell proliferation, and at least in part to a humoral regulatory system. In the present work it has been investigated the role that circulating activities have in the CFU- S stimulation, by means of in vitro and in vivo incubation assays with diffusion chambers. The results show that bone marrow of mice irradiated with 5 Gy produces in vitro diffusible activities capable of stimulating the CFU-S proliferation. As well with this same dose circulating activities are also produced in vivo. In addition we have observed that these activities are only released during the periods of active hemopoietic regeneration that follow irradiation with moderate doses (1.5 and 5 Gy). In another set of experiments we saw that the stimulating activities are also detected in serum of mice irradiated with 5 Gy. These serum activities modify the proliferative state of very primitive precursors (12 d CFU-S). When the serum activities are added to long term bone marrow cultures the CFU-S) are also stimulated to proliferate. Finally, we observed that the radiation-induced serum activities stimulate the proliferation of bone marrow CFU-S when injected into normal mice, suggesting that such activities are involved in the regulation of CFU-S proliferation. (Author)

  6. Probing Androgen Receptor Signaling in Circulating Tumor Cells in Prostate Cancer

    Science.gov (United States)

    2014-07-01

    searched PubMed for English - language full-text manuscripts and abstracts published up to November 2013. The search terms used, alone and in various...cell morphology , and intracellular signalling events in response to therapy. Technologies for the detection of CTCs The presence of CTCs in a...CellSearch® images.44 This automated algor ithm has also been used to extract data on the morphological features of CTCs, including cell size, roundness

  7. Chronic activation profile of circulating CD8+ T cells in Sézary syndrome.

    Science.gov (United States)

    Torrealba, Marina Passos; Manfrere, Kelly Cristina; Miyashiro, Denis R; Lima, Josenilson F; de M Oliveira, Luana; Pereira, Nátalli Z; Cury-Martins, Jade; Pereira, Juliana; Duarte, Alberto J S; Sato, Maria N; Sanches, José A

    2018-01-09

    Sézary syndrome (SS) is a leukemic variant of cutaneous T cell lymphoma (CTCL), and the neoplastic CD4+ T cells of SS patients undergo intense clonal proliferation. Although Sézary cells have been studied extensively, studies on adaptive immunity regarding CD8+T cells are scarce. This study aimed to investigate activation marker expression in CD8+ T cells according to the differentiation stages and IL-7/IL7Rα axis responses of patients with SS. Moreover, this study aimed to verify the soluble forms of CD38, sCD127 and IL-7 in serum. Although the SS patients of our cohort had reduced numbers of CD8+ T cells, they exhibited higher percentages of CD8+CD38+ T cells, mainly effector/memory CD8+ T cells, than the control group. In contrast, down-regulated expression of the activation markers CD127/IL-7R and CD26 was found in the CD8+ T cells of SS patients. High serum levels of sCD38 and sCD127 and scarce serum levels of IL-7 were detected, emphasizing the immune activation status of SS patients. Moreover, CD8+ T cells from SS patients exhibited IL-7 unresponsiveness to STAT5 phosphorylation and Bcl-2 expression, and IL-7 priming partially restored IFNγ production. Our findings showed a chronic activation profile of CD8+ T cells, as an attenuated cytotoxic profile and impaired IL-7 responsiveness was observed, suggesting chronic activation status of CD8+ T cells in SS patients.

  8. A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay.

    Science.gov (United States)

    Magbanua, Mark Jesus M; Pugia, Michael; Lee, Jin Sun; Jabon, Marc; Wang, Victoria; Gubens, Matthew; Marfurt, Karen; Pence, Julia; Sidhu, Harwinder; Uzgiris, Arejas; Rugo, Hope S; Park, John W

    2015-01-01

    Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

  9. A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay.

    Directory of Open Access Journals (Sweden)

    Mark Jesus M Magbanua

    Full Text Available Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19 and lung cancer patients (n = 21, and healthy controls (n = 30 using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs, circulating mesenchymal cells (CMCs, putative circulating stem cells (CSCs, and circulating endothelial cells (CECs. Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85% and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001. Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03. Fifty-three percent (53% of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001. In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47. Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

  10. Induction of Robust B Cell Responses after Influenza mRNA Vaccination Is Accompanied by Circulating Hemagglutinin-Specific ICOS+ PD-1+ CXCR3+ T Follicular Helper Cells

    Directory of Open Access Journals (Sweden)

    Gustaf Lindgren

    2017-11-01

    Full Text Available Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP in non-human primates. While both intradermal (ID and intramuscular (IM administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.

  11. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.

    Science.gov (United States)

    O'Brien, Christine M; Rood, Kyle D; Bhattacharyya, Kiran; DeSouza, Thiago; Sengupta, Shramik; Gupta, Sagar K; Mosley, Jeffrey D; Goldschmidt, Benjamin S; Sharma, Nikhilesh; Viator, John A

    2012-06-01

    Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

  12. Evaluation of droplet digital PCR for quantification of residual leucocytes in red blood cell concentrates.

    Science.gov (United States)

    Doescher, A; Loges, U; Petershofen, E K; Müller, T H

    2017-11-01

    Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR. Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control. © 2017 International Society of Blood Transfusion.

  13. Occurring of In Vitro Functional Vasculogenic Pericytes from Human Circulating Early Endothelial Precursor Cell Culture

    Directory of Open Access Journals (Sweden)

    Silvia Cantoni

    2015-01-01

    Full Text Available Pericytes are periendothelial cells of the microcirculation which contribute to tissue homeostasis and hemostasis by regulating microvascular morphogenesis and stability. Because of their multipotential ex vivo differentiation capabilities, pericytes are becoming very interesting in regenerative medicine field. Several studies address this issue by attempting to isolate pericyte/mesenchymal-like cells from peripheral blood; however the origin of these cells and their culture conditions are still debated. Here we showed that early Endothelial Progenitor