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Sample records for cytometric granulocyte immunofluorescence

  1. A Novel Tool for High-Throughput Screening of Granulocyte-Specific Antibodies Using the Automated Flow Cytometric Granulocyte Immunofluorescence Test (Flow-GIFT

    Directory of Open Access Journals (Sweden)

    Xuan Duc Nguyen

    2011-01-01

    Full Text Available Transfusion-related acute lung injury (TRALI is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT and granulocyte agglutination test (GAT were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti—HNA 3a, n = 3; anti—HNA-1b, n = 1 and GAT (anti—HNA-2a, n = 1. The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of

  2. A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

    Science.gov (United States)

    Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

    2011-02-03

    Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

  3. Immunofluorescence

    International Nuclear Information System (INIS)

    Bongertz, V.; Castro, B.G.

    1990-01-01

    The advantages of the immunofluorescence assay (IFA) that allowing the specific 'in situ' localization of the antigen-antibody interaction, of paramount importance when complex antigenic preparations, such as for instance tissue sections, are studied. (L.M.J.) [pt

  4. Use of a Granulocyte Immunofluorescence Assay Designed for Humans for Detection of Antineutrophil Cytoplasmic Antibodies in Dogs with Chronic Enteropathies.

    Science.gov (United States)

    Florey, J; Viall, A; Streu, S; DiMuro, V; Riddle, A; Kirk, J; Perazzotti, L; Affeldt, K; Wagner, R; Vaden, S; Harris, T; Allenspach, K

    2017-07-01

    Perinuclear antineutrophil cytoplasmic antibodies (pANCA) previously have been shown to be serum markers in dogs with chronic enteropathies, with dogs that have food-responsive disease (FRD) having higher frequencies of seropositivity than dogs with steroid-responsive disease (SRD). The indirect immunofluorescence (IIF) assay used in previous publications is time-consuming to perform, with low interobserver agreement. We hypothesized that a commercially available granulocyte IIF assay designed for humans could be used to detect perinuclear antineutrophil cytoplasmic antibodies in dogs. Forty-four dogs with FRD, 20 dogs with SRD, 20 control dogs, and 38 soft-coated wheaten terrier (SCWT) or SCWT-cross dogs. A granulocyte assay designed for humans was used to detect pANCA, cANCA, and antinuclear antibodies (ANA), as well as antibodies against proteinase-3 protein (PR-3) and myeloperoxidase protein (MPO) in archived serum samples. Sensitivity of the granulocyte assay to predict FRD in dogs was 0.61 (95% confidence interval (CI), 0.45, 0.75), and specificity was 1.00 (95% CI, 0.91, 1.00). A significant association was identified between positive pANCA or cANCA result and diagnosis of FRD (P < 0.0001). Agreement between the two assays to detect ANCA in the same serum samples from SCWT with protein-losing enteropathy/protein-losing nephropathy (PLE/PLN) was substantial (kappa, 0.77; 95% CI, 0.53, 1.00). Eight ANCA-positive cases were positive for MPO or PR-3 antibodies. The granulocyte immunofluorescence assay used in our pilot study was easy and quick to perform. Agreement with the previously published method was good. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  5. Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

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    Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

    2017-11-01

    Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

  6. Granulocyte kinetics

    International Nuclear Information System (INIS)

    Peters, A.M.; Lavender, J.P.; Saverymuttu, S.H.

    1985-01-01

    By using density gradient materials enriched with autologous plasma, the authors have been able to isolate granulocutes from other cellular elements and label them with In-111 without separation from a plasma environment. The kinetic behavior of these cells suggests that phenomena attributed to granulocyte activation are greatly reduced by this labeling. Here, they review their study of granulocyte kinetics in health and disease in hope of quantifying sites of margination and identifying principal sites of destruction. The three principle headings of the paper are distribution, life-span, and destruction

  7. Granulocytic sarcoma.

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    Hutchison, R E; Kurec, A S; Davey, F R

    1990-12-01

    Granulocytic sarcoma is a variant presentation of acute myeloblastic leukemia, occurring in extramedullary locations. It is uncommon, but it may occur at any site and at any age, which necessitates its inclusion in the differential diagnosis of all undifferentiated tumors. Histology, touch-imprint cytology, cytochemistry, immunocytochemistry, electron microscopy, and molecular studies all contribute to the diagnosis.

  8. Cytometric approaches to biological dosimetry

    International Nuclear Information System (INIS)

    Burger, G.

    1983-01-01

    Automatic cytometric techniques for detecting chromosomal aberrations are being tested but will not be used in routine examinations for some time to come. Automatic micronuclei counts are more promising but not sufficiently sensitive in the low dose range ( [de

  9. Cytometric analysis of irradiation damaged chromosomes

    International Nuclear Information System (INIS)

    Wilder, M.E.; Raju, M.R.

    1982-01-01

    Irradiation of cells in interphase results in dose-dependent damage to DNA which is discernable by flow-cytometric analysis of chromosomes. The quantity (and possibly the quality) of chromosomal changes is different in survival-matched doses of x and α irradiation. It may, therefore, be possible to use these methods for analysis of dose and type of exposure in unknown cases

  10. Irradiation for conjunctival granulocytic sarcoma

    International Nuclear Information System (INIS)

    Fleckenstein, K.; Geinitz, H.; Grosu, A.; Molls, M.; Goetze, K.; Werner, M.

    2003-01-01

    Case History and Findings: A 73-year-old woman with a history of myeloproliferative syndrome (MPS) presented with bilateral chemosis, redness and burning of the eyes. The ocular motility was severely impaired. Ophthalmological examination revealed markedly distended conjunctivas on both sides. Biopsy disclosed conjunctival granulocytic sarcoma as an initial symptom of acute myelogenous leukemia (AML). Diagnosis was confirmed by peripheral blood smear and bone marrow aspiration. Treatment and Outcome: The orbital tumor disappeared completely after local external beam irradiation with a total dose of 30 Gy and no further orbital recurrence occurred. With chemotherapy following irradiation transient hematological remission was achieved. 5 months after diagnosis the patient died of respiratory failure following atypical pneumonia as a consequence of her underlying disorder. Conclusion: Detection of orbital granulocytic sarcoma, even in the absence of typical leukemic symptoms is of practical importance, because treatment with irradiation can lead to stabilization or improvement in the patient's vision. (orig.)

  11. Biostatistical analysis of quantitative immunofluorescence microscopy images.

    Science.gov (United States)

    Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

    2016-12-01

    Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  12. Testicular granulocytic sarcoma without systemic leukemia

    NARCIS (Netherlands)

    Lagerveld, B. W.; Wauters, C. A. P.; Karthaus, H. F. M.

    2005-01-01

    This case report describes a unilateral testicular granulocytic sarcoma or chloroma. Because of the relatively immature nature of the tumor cells, the histological diagnosis can be difficult. Granulocytic sarcomas are well known in patients with systemic leukemia and can sometimes precede a systemic

  13. Epidermis area detection for immunofluorescence microscopy

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    Dovganich, Andrey; Krylov, Andrey; Nasonov, Andrey; Makhneva, Natalia

    2018-04-01

    We propose a novel image segmentation method for immunofluorescence microscopy images of skin tissue for the diagnosis of various skin diseases. The segmentation is based on machine learning algorithms. The feature vector is filled by three groups of features: statistical features, Laws' texture energy measures and local binary patterns. The images are preprocessed for better learning. Different machine learning algorithms have been used and the best results have been obtained with random forest algorithm. We use the proposed method to detect the epidermis region as a part of pemphigus diagnosis system.

  14. Direct immunofluorescence for the diagnosis of legionellosis

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    David JM Haldane

    1993-01-01

    Full Text Available Culture and direct immunofluorescent microscopy (DFA results for Legionella pneumophila were reviewed over a two-year period. In the first year, a positive result was defined as having at least one morphologically typical fluorescing organism. In the second year, a positive was defined as at least five typical fluorescing organisms. Despite these stricter criteria and other measures to reduce the possibility of reagent contamination, there was no statistically significant difference in the sensitivity or specificity of the DFA in the two years for sputa, deep specimens or overall. Of 37 sputum specimens from infected patients, 16 were positive on DFA. Thirty-two of 38 positive patients were detected by sputum culture. DFA can provide rapid diagnostic information but cannot be used to rule out the diagnosis. Sputum is a useful specimen for the initial laboratory investigation of patients with legionellosis.

  15. An antigen shared by human granulocytes, monocytes, marrow granulocyte precursors and leukemic blasts.

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    Shumak, K H; Rachkewich, R A

    1983-01-01

    An antibody to human granulocytes was raised in rabbits by immunization with granulocytes pretreated with rabbit antibody to contaminating antigens. The antibody reacted not only with granulocytes but also with monocytes and bone marrow granulocyte precursors including colony-forming units in culture (CFU-C). In tests with leukemic cells, the antibody reacted with blasts from most (8 of 9) patients with acute myelomonoblastic leukemia and from some patients with acute myeloblastic leukemia, morphologically undifferentiated acute leukemia and chronic myelogenous leukemia in blast crisis. The antibody did not react with blasts from patients with acute lymphoblastic leukemia nor with leukemic cells from patients with chronic lymphocytic leukemia.

  16. Flow cytometric characterization of cerebrospinal fluid cells.

    Science.gov (United States)

    de Graaf, Marieke T; de Jongste, Arjen H C; Kraan, Jaco; Boonstra, Joke G; Sillevis Smitt, Peter A E; Gratama, Jan W

    2011-09-01

    Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately. Copyright © 2011 International Clinical Cytometry Society.

  17. An Automatic Indirect Immunofluorescence Cell Segmentation System

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    Yung-Kuan Chan

    2014-01-01

    Full Text Available Indirect immunofluorescence (IIF with HEp-2 cells has been used for the detection of antinuclear autoantibodies (ANA in systemic autoimmune diseases. The ANA testing allows us to scan a broad range of autoantibody entities and to describe them by distinct fluorescence patterns. Automatic inspection for fluorescence patterns in an IIF image can assist physicians, without relevant experience, in making correct diagnosis. How to segment the cells from an IIF image is essential in developing an automatic inspection system for ANA testing. This paper focuses on the cell detection and segmentation; an efficient method is proposed for automatically detecting the cells with fluorescence pattern in an IIF image. Cell culture is a process in which cells grow under control. Cell counting technology plays an important role in measuring the cell density in a culture tank. Moreover, assessing medium suitability, determining population doubling times, and monitoring cell growth in cultures all require a means of quantifying cell population. The proposed method also can be used to count the cells from an image taken under a fluorescence microscope.

  18. Granulocyte-mobilized bone marrow.

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    Arcese, William; De Angelis, Gottardo; Cerretti, Raffaella

    2012-11-01

    In the last few years, mobilized peripheral blood has overcome bone marrow as a graft source, but, despite the evidence of a more rapid engraftment, the incidence of chronic graft-versus-host disease is significantly higher with, consequently, more transplant-related mortality on the long follow-up. Overall, the posttransplant outcome of mobilized peripheral blood recipients is similar to that of patients who are bone marrow grafted. More recently, the use of bone marrow after granulocyte colony-stimulating factor (G-CSF) donor priming has been introduced in the transplant practice. Herein, we review biological acquisitions and clinical results on the use of G-CSF-primed bone marrow as a source of hematopoietic stem cells (HSC) for allogeneic stem cell transplantation. G-CSF the increases the HSC compartment and exerts an intense immunoregulatory effect on marrow T-cells resulting in the shift from Th1 to Th2 phenotype with higher production of anti-inflammatory cytokines. The potential advantages of these biological effects have been translated in the clinical practice by using G-CSF primed unmanipulated bone marrow in the setting of transplant from human leukocyte antigen (HLA)-haploidentical donor with highly encouraging results. For patients lacking an HLA-identical sibling, the transplant of G-CSF primed unmanipulated bone marrow from a haploidentical donor combined with an intense in-vivo immunosuppression is a valid alternative achieving results that are well comparable with those reported for umbilical cord blood, HLA-matched unrelated peripheral blood/bone marrow or T-cell-depleted haploidentical transplant.

  19. Hemopathologic consequences of protracted gamma irradiation: alterations in granulocyte reserves and granulocyte mobilization

    International Nuclear Information System (INIS)

    Seed, T.M.; Cullen, S.M.; Kaspar, L.V.; Tolle, D.V.; Fritz, T.E.

    1980-01-01

    Aplastic anemia and myelogenous leukemia are prominent pathologic effects in beagles exposed to continuous, daily, low-dose gamma irradiation. In the present work, granulocyte reserves and related mobilization functions have been sequentially assessed by the endotoxin stress assay during the preclinical and clinical phases of these hemopoietic disorders. Characteristic patterns of granulocyte reserve mobilization are described that reflect given stages of pathologic progression. For radiation-induced leukemia, a five-stage pattern has been proposed. In contrast, a simple pattern of progressive, time-dependent contraction of granulocyte reserves and mobilization capacity was noted in the development of terminal aplastic anemia. Early preclinical phases of radiation-induced leukemia appear to involve an extensive depletion of the granulocyte reserves (phase I) during the first approx. 200 days of exposure followed by a partial renewal of the reserves and associated mobilization functions between approx. 200 and 400 days (phase II). Sustained, subnormal granulocyte mobilizations (phase III) following endotoxin stress typify the responses of dogs during the intermediate phase, whereas late preclinical, preleukemic stages (phase IV) are characterized by a further expansion of the reserves and in the mobilization capacities, particularly of the less mature granulocytes. Such late alterations in the pattern of granulocyte mobilization, together with other noted cellular aberrancies in the peripheral blood and marrow, appear to indicate leukemia (phase V) onset

  20. The redistribution of granulocytes following E. coli endotoxin induced sepsis

    DEFF Research Database (Denmark)

    Toft, P; Lillevang, S T; Tønnesen, Else Kirstine

    1994-01-01

    Infusion of endotoxin elicits granulocytopenia followed by increased numbers of granulocytes in peripheral blood. The purpose of this study was to investigate the redistribution and sequestration of granulocytes in the tissues following E. coli endotoxin induced sepsis. From 16 rabbits granulocytes...

  1. Antibodies to UV irradiated DNA: the monitoring of DNA damage by ELISA and indirect immunofluorescence

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    Wani, A A; Gibson-D' Ambrosio, R E; D' Ambrosio, S M [Ohio State Univ., Columbus (USA). Dept. of Radiology

    1984-10-01

    The enzyme-linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV-irradiated single-stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC). Fifty per cent of the maximum antibody binding was observed at a 1-5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody, whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the UV dose to DNA and the concentration of antigen immobilized on the plate. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establishes that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated cells.

  2. Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis ▿ †

    Science.gov (United States)

    Keserue, Hans-Anton; Füchslin, Hans Peter; Egli, Thomas

    2011-01-01

    Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated. PMID:21685159

  3. Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Solis, E.; Mackey, S.T.; Socher, S.; Chowdhury, S.; Wu, F.P.; Kaye, M.P.

    1985-01-01

    A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of the femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111 In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into the thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation

  4. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... daughter cells, leading to a characteristic flow cytometric profile where a ... cell recognition without any impact on bone marrow hemato- ... cells of various cancer cells that load CFSE concentration ... (B) Target cells (R1) were further analyzed in an FL1/FL3 dot plot, ..... hematopoietic cell transplantation.

  5. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  6. Estimation of antibodies to human cytomegalovirus by immunofluorescence and radioimmunoassay

    International Nuclear Information System (INIS)

    Jankowski, M.; Gut, W.; Nawrocka, E.

    1980-01-01

    The 125 I labelled IgG fraction against rabbit IgG of goat origin was employed for the detection of CMV IgG and IgM antibodies in the double indirect radioimmunoassay. The results were compared with those obtained in complement fixation, indirect immunofluorescence and anti-complement immunofluorescence tests. The application of labelled anti-fc antisera, instead of antisera against whole IgG in the tests for detection of specific CMV IgG antibody resulted in increased sensitivity of radioimmunoassay and reduction of non-specific cytoplasmatic reactions in preparations stained by indirect immunofluorescence. The absorption of sera with protein A rich staphylococci and aggregates to immunoglobulin eliminated unwanted secondary IgM staining caused by rheumatoid factors both in indirect immunofluorescence and radioimmunoassay tests for CMV antibodies. (author)

  7. Extracellular Trap Formation in Response to Trypanosoma cruzi Infection in Granulocytes Isolated From Dogs and Common Opossums, Natural Reservoir Hosts

    Directory of Open Access Journals (Sweden)

    Nicole de Buhr

    2018-05-01

    Full Text Available Granulocytes mediate the first line of defense against infectious diseases in humans as well as animals and they are well known as multitasking cells. They can mediate antimicrobial activity by different strategies depending on the pathogen they encounter. Besides phagocytosis, a key strategy against extracellular pathogens is the formation of extracellular traps (ETs. Those ETs mainly consist of DNA decorated with antimicrobial components and mediate entrapment of various pathogens. In the last years, various studies described ET formation as response to bacteria, viruses and parasites e.g., Trypanosma (T. cruzi. Nevertheless, it is not fully understood, if ET formation helps the immune system to eliminate intracellular parasites. The goal of this study was to analyze ET formation in response to the intracellular parasite Trypanosma (T. cruzi by granulocytes derived from animals that serve as natural reservoir. Thus, we investigated the ET formation in two T. cruzi reservoirs, namely dogs as domestic animal and common opossums (Didelphis marsupialis as wild animal. Granulocytes were harvested from fresh blood by density gradient centrifugation and afterwards incubated with T. cruzi. We conducted the analysis by determination of free DNA and immunofluorescence microscopy. Using both methods, we show that T. cruzi efficiently induces ET formation in granulocytes derived from common opossum as well as dog blood. Most ETs from both animal species as response to T. cruzi are decorated with the protease neutrophil elastase. Since T. cruzi is well known to circulate over years in both analyzed animals as reservoirs, it may be assumed that T. cruzi efficiently evades ET-mediated killing in those animals. Therefore, ETs may not play a major role in efficient elimination of the pathogen from the blood of dogs or common opossums as T. cruzi survives in niches of their body. The characterization of granulocytes in various animals and humans may be helpful

  8. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

    Science.gov (United States)

    Yu, Yen-Rei A.; O’Koren, Emily G.; Hotten, Danielle F.; Kan, Matthew J.; Kopin, David; Nelson, Erik R.; Que, Loretta; Gunn, Michael D.

    2016-01-01

    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions. PMID:26938654

  9. (TH) diazepam binding to human granulocytes

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    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  10. MRI of perineural extramedullary granulocytic sarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Graham, A. [Rehabilitation Medicine, Hunters Moor Neurological Rehabilitation Centre, Newcastle-Upon-Tyne (United Kingdom); Hodgson, T. [Neuroradiology Dept., Royal Hallamshire Hospital, Sheffield (United Kingdom); Jacubowski, J. [Neurosurgical Dept., Royal Hallamshire Hospital, Sheffield (United Kingdom); Norfolk, D. [Haematology Department, Leeds General Infirmary, Leeds LS1 3EX (United Kingdom); Smith, C. [Pathology Dept., Royal Hallamshire Hospital, Sheffield (United Kingdom)

    2001-06-01

    Granulocytic sarcoma is an extramedullary solid tumour consisting of myelogenous leukaemic blast cells, usually seen in acute myeloid leukaemia and less commonly in patients with chronic myeloid leukaemia or myeloproliferative disorders. Blast cells have a predilection for periosteal and perineural regions and rarely precede evidence of systemic disease. We present two patients, aleukaemic on peripheral blood counts, both at presentation and during subsequent treatment. We present the MRI features of this rare but important condition. (orig.)

  11. Flow cytometric fingerprinting for microbial strain discrimination and physiological characterization.

    Science.gov (United States)

    Buysschaert, Benjamin; Kerckhof, Frederiek-Maarten; Vandamme, Peter; De Baets, Bernard; Boon, Nico

    2018-02-01

    The analysis of microbial populations is fundamental, not only for developing a deeper understanding of microbial communities but also for their engineering in biotechnological applications. Many methods have been developed to study their characteristics and over the last few decades, molecular analysis tools, such as DNA sequencing, have been used with considerable success to identify the composition of microbial populations. Recently, flow cytometric fingerprinting is emerging as a promising and powerful method to analyze bacterial populations. So far, these methods have primarily been used to observe shifts in the composition of microbial communities of natural samples. In this article, we apply a flow cytometric fingerprinting method to discriminate among 29 Lactobacillus strains. Our results indicate that it is possible to discriminate among 27 Lactobacillus strains by staining with SYBR green I and that the discriminatory power can be increased by combined SYBR green I and propidium iodide staining. Furthermore, we illustrate the impact of physiological changes on the fingerprinting method by demonstrating how flow cytometric fingerprinting is able to discriminate the different growth phases of a microbial culture. The sensitivity of the method is assessed by its ability to detect changes in the relative abundance of a mix of polystyrene beads down to 1.2%. When a mix of bacteria was used, the sensitivity was as between 1.2% and 5%. The presented data demonstrate that flow cytometric fingerprinting is a sensitive and reproducible technique with the potential to be applied as a method for the dereplication of bacterial isolates. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  12. Immunofluorescence detection of pea protein in meat products.

    Science.gov (United States)

    Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

    2016-08-01

    In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

  13. Influence of UV-radiation on granulocytic phagocytosis in vitro

    International Nuclear Information System (INIS)

    Walther, T.; Rytter, M.; Gast, W.; Haustein, U.F.

    1987-01-01

    The influence of UV radiation on the vitality, the performance of phagocytosis and the ability to reduce nitro-blue tetrazolium test (NBT) by human granulocytes was investigated in vitro. Already by low doses of UVA (8% UVB) the percentage of phagocytizing granulocytes was decreased more distinctly than their cell vitality. The number of ingested Candida albicans particles was 4.5 particles per granulocyte in the controls. It was reduced to about 1.4 particles per cell by UV radiation independent of the dosis applied. On the other hand the ability of granulocytes to reduce NBT intracellularly remained completely unchanged. (author)

  14. Granulocyte migration in uncomplicated intestinal anastomosis in man

    Energy Technology Data Exchange (ETDEWEB)

    Keshavarzian, A.; Gibson, R.; Guest, J.; Spencer, J.; Lavender, J.P.; Hodgson, H.J.

    1986-03-01

    We have investigated the presence, duration, and clinical significance of granulocyte accumulation, using indium-111 granulocyte scanning, in patients following uncomplicated intestinal anastomosis. Eight patients underwent intestinal resection and anastomosis (right hemicolectomy, 5; sigmoid colectomy, 2; ileal resection, 1) for carcinoma, angiodysplasia, or perforation. All patients had an uneventful postoperative course, with no evidence of any leakage or infection. Indium-111 granulocyte scan and abdominal ultrasound were performed 7-20 days (12 +/- 4.7 means +/- SD) following surgery. Indium-111 granulocyte scan showed the presence of labeled granulocytes at the site of anastomosis in all patients. In three of eight, cells subsequently passed into the lumen of the bowel. In contrast, granulocytes were not visualized along the abdominal incision. Thus, in contrast to skin wounds, granulocytes continue migrating into the intestinal wall in areas of anastomosis for at least up to 20 days following surgical trauma. They may play a significant role both in healing the anastomosis and in preventing systemic bacterial infection. Moreover, indium-111 granulocyte scans following intestinal surgery should be interpreted with care, and the presence of labeled granulocytes around anastomoses does not necessarily indicate abscess formation.

  15. Granulocyte migration in uncomplicated intestinal anastomosis in man

    International Nuclear Information System (INIS)

    Keshavarzian, A.; Gibson, R.; Guest, J.; Spencer, J.; Lavender, J.P.; Hodgson, H.J.

    1986-01-01

    We have investigated the presence, duration, and clinical significance of granulocyte accumulation, using indium-111 granulocyte scanning, in patients following uncomplicated intestinal anastomosis. Eight patients underwent intestinal resection and anastomosis (right hemicolectomy, 5; sigmoid colectomy, 2; ileal resection, 1) for carcinoma, angiodysplasia, or perforation. All patients had an uneventful postoperative course, with no evidence of any leakage or infection. Indium-111 granulocyte scan and abdominal ultrasound were performed 7-20 days (12 +/- 4.7 means +/- SD) following surgery. Indium-111 granulocyte scan showed the presence of labeled granulocytes at the site of anastomosis in all patients. In three of eight, cells subsequently passed into the lumen of the bowel. In contrast, granulocytes were not visualized along the abdominal incision. Thus, in contrast to skin wounds, granulocytes continue migrating into the intestinal wall in areas of anastomosis for at least up to 20 days following surgical trauma. They may play a significant role both in healing the anastomosis and in preventing systemic bacterial infection. Moreover, indium-111 granulocyte scans following intestinal surgery should be interpreted with care, and the presence of labeled granulocytes around anastomoses does not necessarily indicate abscess formation

  16. Granulocytic sarcoma: a rare cause of sciatica.

    Science.gov (United States)

    Valsamis, Epaminondas Markos; Glover, Thomas Edward

    2017-02-15

    We describe a case report of a man aged 56 years with a 4-month history of right-sided sciatica-type pain with subclinical disc prolapse evident on MRI. Worsening pain together with the appearance of a tender mass in his right buttock prompted further imaging, which demonstrated an infiltrative mass engulfing the lumbosacral plexus. This was later shown to be a granulocytic sarcoma on biopsy. Intervertebral disc herniation can be an incidental finding and is not always the cause of sciatica. 2017 BMJ Publishing Group Ltd.

  17. Role of direct immunofluorescence in the diagnosis of glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Archana C Buch

    2015-01-01

    Full Text Available Background: Immunofluorescence microscopy is a vital tool for the diagnosis of glomerular diseases. This study was carried out to study patterns of glomerulonephritis (GN and to record the sensitivity of direct immunofluorescence (DIF in renal lesions. The DIF findings were correlated with clinical and histopathology findings and discrepancies were analyzed. Materials and Methods: The cross-sectional analytical study was conducted during the period July 2011 to July 2013 at a tertiary care Hospital, Department of Pathology. A total of 75 renal biopsies were received for routine and immunofluorescence studies in which histopathology and clinical data were reviewed and analyzed. Results: The sensitivity of DIF was 87.9% and specificity was 70.5%. The maximum number of cases were seen in the age group 41-50 years. The pattern of GN by DIF was minimal change disease (MCD in 24%, IgA nephropathy in 13%, focal segmental glomerulosclerosis in 9% and membranoproliferative glomerulonephritis in 8% of the cases. Twelve histopathologically proven cases of GN were negative on DIF. One case of MCD on histopathology was diagnosed as IgM nephropathy based on DIF. Conclusion: Direct immunofluorescence forms an important diagnostic tool in reaching the exact diagnosis in various types of GN presenting with overlapping clinical and histomorphological features.

  18. Acquired agranulocytosis with granulocyte specific cytotoxic autoantibody.

    Science.gov (United States)

    Blaschke, J; Goeken, N E; Thompson, J S; Dick, F R; Gingrich, R D

    1979-05-01

    Multiple infections and severe neutropenia were found in a previously healthy 29 year old man with no history of similar syndromes in the family, drug ingestion or exposure to environmental toxins. There was no evidence at the time of presentation of diseases previously associated with agranulocytosis (e.g., neoplasia, thyrotoxicosis, chronic infection, collagen-vascular disease or leukoagglutinating antibody). His serum contained a nonagglutinating, complement-dependent, cytotoxic antibody, however, reactive with peripheral blood granulocytes from 35 per cent of normal donors. The neutropenia was not affected by steroids but resolved promptly after splenectomy. Microscopic examination of the spleen revealed ingestion of polymorphonuclear leukocytes by splenic macrophages. Family studies indicated that the target antigen was non-HLA and that the antibody was not absorbed by lymphocytes or platelets. We conclude that the agranulocytosis was autoimmune in origin and suggest that similar myeloid-specific immune responses could influence granulocyte tranfusion and bone marrow transplantation by alloimmune "rejection" that would not be avoided by matching only for HLA specificities.

  19. The redistribution of granulocytes following E. coli endotoxin induced sepsis

    DEFF Research Database (Denmark)

    Toft, P; Lillevang, S T; Tønnesen, Else Kirstine

    1994-01-01

    Infusion of endotoxin elicits granulocytopenia followed by increased numbers of granulocytes in peripheral blood. The purpose of this study was to investigate the redistribution and sequestration of granulocytes in the tissues following E. coli endotoxin induced sepsis. From 16 rabbits granulocytes...... were isolated, labelled with Indium and reinjected intravenously. Eight rabbits received an infusion of E. coli endotoxin 2 micrograms kg-1 while eight received isotonic saline. The redistribution of granulocytes was imaged with a gamma camera and calculated with a connected computer before and 2 and 6...... hours after infusion of endotoxin or saline. Serum cortisol and interleukin-1 beta were measured. In another seven rabbits, respiratory burst activity and degranulation of granulocytes were measured prior to and from 5 min to 6 hours after infusion of E. coli endotoxin 2 micrograms kg-1 BW. Following...

  20. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  1. Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

    International Nuclear Information System (INIS)

    Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

    1988-01-01

    The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

  2. Cytometric analysis of mammalian sperm for induced morphologic and DNA content errors

    International Nuclear Information System (INIS)

    Pinkel, D.

    1983-01-01

    Some flow-cytometric and image analysis procedures under development for quantitative analysis of sperm morphology are reviewed. The results of flow-cytometric DNA-content measurements on sperm from radiation exposed mice are also summarized, the results related to the available cytological information, and their potential dosimetric sensitivity discussed

  3. Conservation of myeloid surface antigens on primate granulocytes.

    Science.gov (United States)

    Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

    1983-02-01

    Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

  4. Autologous 111In-oxine-labeled granulocytes in Yersinia infections

    International Nuclear Information System (INIS)

    Becker, W.; Boerner, W.; Fischbach, W.

    1985-01-01

    Autologous 111 In-oxine-labeled granulocytes have proved to be valuable for the localization of inflammatory bowel diseases, especially Crohn's disease and ulcerative colitis. Other rare inflammatory bowel diseases also yield positive 111 In scans. One case of Yersinia infection of the terminal ileum (Yersinia enterocolitica) showing an accumulation of 111 In-oxine-labeled granulocytes 0.5, 4, and 24 h after the reinjection of the labeled cells is described. The 4-day fecal excretion of 111 In-oxine granulocytes showed a slight inflammatory activity of the terminal ileum. One negative scan is reported in a cotrimoxazole-treated patient with Yersinia infection. (orig.)

  5. Granulocytic Sarcoma of the Stomach Presenting as Dysphagia during Pregnancy

    Directory of Open Access Journals (Sweden)

    Anuradha Sekaran

    2011-01-01

    Full Text Available Granulocytic sarcoma also known as extramedullary myeloid sarcoma or chloroma is an uncommon manifestation of leukemia and presents as a deposit of leukemic cells outside the bone marrow. We report a case of a twenty-five-year-old pregnant woman who presented with progressive dysphagia and recurrent postprandial vomiting. Upper GI endoscopy had shown large flat laterally spread nodular lesions in the cardia and proximal body of stomach. Biopsies from the gastric lesion showed granulocytic sarcoma of the stomach. Concurrent peripheral and bone marrow picture was suggestive of acute myeloid leukemia (AML–M4. There is limited reported literature on granulocytic sarcoma of the stomach. Concurrent gastric granulocytic sarcoma involving cardia and AML in pregnancy has not been reported till date.

  6. Hungry granulocyte: its fate and regulation of production

    International Nuclear Information System (INIS)

    Cronkite, E.P.

    1978-01-01

    The granulocyte, a phagocytic anti-1 bacterial defense cell, is discussed. Its production, the kinetics of its proliferation, the regulation of its production, and its loss from the blood are reviewed

  7. Indium-111 granulocyte scintigraphy in inflammatory bowel disease

    International Nuclear Information System (INIS)

    Devillers, A.; Moisan, A.; Heresbach, D.; Darnault, P.; Bretagne, J.F.

    1996-01-01

    The present paper reports our experience since 1963 concerning 111-indium labeled autologous granulocytes scanning in the assessment of inflammatory bowel diseases and in the assessment of activity in Crohn's disease and ulcerative colitis. (authors). 94 refs., 3 figs

  8. Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

    Science.gov (United States)

    Dillinger, Stefan; Németh, Attila

    2016-01-01

    The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

  9. [Laboratory diagnosis of genital herpes--direct immunofluorescence method].

    Science.gov (United States)

    Majewska, Anna; Romejko-Wolniewicz, Ewa; Zareba-Szczudlik, Julia; Kilijańczyk, Marek; Gajewska, Małgorzata; Młynarczyk, Grazyna

    2013-07-01

    Aim of the study was to determine clinical usefulness of direct immunofluorescence method in the laboratory diagnosis of genital herpes in women. Overall 187 anogenital swabs were collected from 120 women. Using a dacron-tipped applicator 83 swabs were collected from women suspected of genital herpes and 104 from patients with no signs of genital infection. All samples were tested using cell culture (Vero cell line) and then direct immunofluorescence method (DIF) for the identification of antigens of herpes simplex viruses: HSV-1 and HSV-2. Characteristic cytopathic effect (CPE), indicative of alphaherpesvirus infection, was observed in 43.4% of cultures with clinical specimens collected from women with suspected genital herpes and in 29.8% of cultures of clinical specimens taken from patients with no clinical symptoms of genital herpes. Herpes simplex viruses were determined in 73 samples by direct immunofluorescence method after amplification of the virus in cell culture. The DIF test confirmed the diagnosis based on the microscopic CPE observation in 85%. In 15% of samples (taken from pregnant women without clinical signs of infection) we reported positive immunofluorescence in the absence of CPE. The frequency of antigen detection was statistically significantly higher in samples that were positive by culture study (chi-square test with Yates's correction, p genital herpes in swabs taken from the vestibule of the vagina and the vulva. However, there was no statistically significant difference in the frequency of detection of Herpes Simplex Virus antigens in specimens from different parts of the genital tract in both groups of women (chi-square test, p > 0.05). In our study HHV-1 was the main causative agent of genital herpes. The growing worldwide prevalence of genital herpes, challenges with the clinical diagnosis, and availability of effective antiviral therapy are the main reasons for a growing interest in rapid, proper laboratory diagnosis of infected

  10. Multivariate analysis of flow cytometric data using decision trees.

    Science.gov (United States)

    Simon, Svenja; Guthke, Reinhard; Kamradt, Thomas; Frey, Oliver

    2012-01-01

    Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called "induction of decision trees" in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees' quality, we used stratified fivefold cross validation and chose the "best" tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.

  11. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Directory of Open Access Journals (Sweden)

    Chang Rae Rho

    Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  12. Diagnostic significance of colloid body deposition in direct immunofluorescence

    Directory of Open Access Journals (Sweden)

    Chularojanamontri Leena

    2010-01-01

    Full Text Available Background: Colloid bodies (CB in direct immunofluorescence (DIF studies are usually found in interface dermatitis. Furthermore, CB can be found in various skin diseases and even in normal skin. Aim: To evaluate the diagnostic value of CB deposits in DIF studies. Methods: From 1996-2007, data from 502 patients where DIF studies showed immunoreactants at CB were enrolled. The definite diagnoses of these patients were based on clinical, histopathological and immunofluorescent findings. The results of DIF studies were analyzed. Results: Immunoreactants at CB were detected in 44.4%, 43.8%, 4.2%, 3.8%, and 2.2% of interface dermatitis, vasculitis, autoimmune vesiculobullous disease, panniculitis, and scleroderma/morphea, respectively. The most common immunoreactant deposit of all diseases was Immunoglobulin M (IgM. Brighter intensity and higher quantity of CB was detected frequently in the group with interface dermatitis. Conclusions: Immunoreactant deposits at CB alone can be found in various diseases but a strong intensity and high quantity favor the diagnosis of interface dermatitis. CB plus dermoepidermal junction (DEJ deposits are more common in interface dermatitis than any other disease. Between lichen planus (LP and discoid lupus erythematosus (DLE, CB alone is more common in LP; whereas, CB plus DEJ and superficial blood vessel (SBV is more common in DLE. The most common pattern in both diseases is CB plus DEJ. The quantity and intensity of CB in LP is higher than in DLE.

  13. Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

    Science.gov (United States)

    Otani, Kei; Niwa, Yuki; Suzuki, Takehiro; Sato, Natsumi; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2018-04-06

    Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Leukocytospermia and sperm preparation - a flow cytometric study

    Directory of Open Access Journals (Sweden)

    Perticarari Sandra

    2009-01-01

    Full Text Available Abstract Background Leukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii to examine the effect of leukocytes on sperm preparation, and (iii to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method. Methods Semen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm. Results Both sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical

  15. 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

  16. Granulocyte colony-stimulating factor and leukemogenesis

    Directory of Open Access Journals (Sweden)

    Lorena Lobo de Figueiredo

    2004-01-01

    Full Text Available THE granulocyte colony-stimulating factor (G-CSF plays an important role in normal granulopoiesis. Its functions are mediated by specific receptors on the surface of responsive cells and, upon ligand binding, several cytoplasmic tyrosine kinases are activated. The cytoplasmic region proximal to the membrane of the G-CSF receptor (G-CSF-R transduces proliferative and survival signals, whereas the distal carboxy-terminal region transduces maturation signals and suppresses the receptor's proliferative signals. Mutations in the G-CSF-R gene resulting in truncation of the carboxy-terminal region have been detected in a subset of patients with severe congenital neutropenia who developed acute myelogenous leukemia (AML. In addition, the AML1-ETO fusion protein, expressed in leukemic cells harboring the t(8;21, disrupt the physiological function of transcription factors such as C/EBPα and C/EBPε, which in turn deregulate G-CSF-R expression. The resulting high levels of G-CSF-R and G-CSF-dependent cell proliferation may be associated with pathogenesis of AML with t(8;21. Moreover, in vitro and in vivo studies demonstrated that G-CSF may act as a co-stimulus augmenting the response of PML-RARα acute promyelocytic leukemia cells to all-trans-retinoic acid treatment. Finally, in the PLZF-RARα acute promyelocytic leukemia transgenic model, G-CSF deficiency suppressed leukemia development. Altogether, these data suggest that the G-CSF signaling pathway may play a role in leukemogenesis.

  17. Radiation Therapy for Chloroma (Granulocytic Sarcoma)

    Energy Technology Data Exchange (ETDEWEB)

    Bakst, Richard; Wolden, Suzanne [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Yahalom, Joachim, E-mail: yahalomj@mskcc.org [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

    2012-04-01

    Objectives: Chloroma (granulocytic sarcoma) is a rare, extramedullary tumor of immature myeloid cells related to acute nonlymphocytic leukemia or myelodysplastic syndrome. Radiation therapy (RT) is often used in the treatment of chloromas; however, modern studies of RT are lacking. We reviewed our experience to analyze treatment response, disease control, and toxicity associated with RT to develop treatment algorithm recommendations for patients with chloroma. Patients and Methods: Thirty-eight patients who underwent treatment for chloromas at our institution between February 1990 and June 2010 were identified and their medical records were reviewed and analyzed. Results: The majority of patients that presented with chloroma at the time of initial leukemia diagnosis (78%) have not received RT because it regressed after initial chemotherapy. Yet most patients that relapsed or remained with chloroma after chemotherapy are in the RT cohort (90%). Thirty-three courses of RT were administered to 22 patients. Radiation subsite breakdown was: 39% head and neck, 24% extremity, 9% spine, 9% brain, 6% genitourinary, 6% breast, 3% pelvis, and 3% genitourinary. Median dose was 20 (6-36) Gy. Kaplan-Meier estimates of progression-free survival and overall survival in the RT cohort were 39% and 43%, respectively, at 5 years. At a median follow-up of 11 months since RT, only 1 patient developed progressive disease at the irradiated site and 4 patients developed chloromas at other sites. RT was well tolerated without significant acute or late effects and provided symptom relief in 95% of cases. Conclusions: The majority of patients with chloromas were referred for RT when there was extramedullary progression, marrow relapse, or rapid symptom relief required. RT resulted in excellent local disease control and palliation of symptoms without significant toxicity. We recommend irradiating chloromas to at least 20 Gy, and propose 24 Gy in 12 fractions as an appropriate regimen.

  18. Granulocytes: New Members of the Antigen-Presenting Cell Family

    Directory of Open Access Journals (Sweden)

    Ang Lin

    2017-12-01

    Full Text Available Granulocytes, the most abundant types of leukocytes, are the first line of defense against pathogen invasion. However, the plasticity and diversity of granulocytes have been increasingly revealed, especially with regard to their versatile functions in orchestrating adaptive immune responses. A substantial body of recent evidence demonstrates that granulocytes can acquire the function as antigen-presenting cells under pathological or inflammatory conditions. In addition, they can acquire surface expression of MHC class II and costimulatory molecules as well as T cell stimulatory behavior when cultured with selected cytokines. The classic view of granulocytes as terminally differentiated, short-lived phagocytes is therefore changing to phenotypically and functionally heterogeneous cells that are engaged in cross-talk with other leukocyte populations and provide an additional link between innate and adaptive immunity. In this brief review, we summarize the current knowledge on the antigen-presenting capacity of granulocyte subsets (neutrophils, eosinophils, and basophils. Underlying mechanisms, relevant physiological significance and potential controversies are also discussed.

  19. Flow cytometric life cycle analysis in cellular radiation biology

    International Nuclear Information System (INIS)

    Wood, J.C.S.

    1982-01-01

    Three approaches to flow cytometric histogram analysis were developed: (1) differential histogram analysis, (2) DNA histogram analysis, and (3) multiparameter data analysis. These techniques were applied to an important unresolved problem in radiation biology. The initial responses to irradiation of a mammalian cell which occur during the first two cell cycles following the irradiation are of considerable interest to the radiation biologist. During the first two post-irradiation cell cycles, cells which ultimately will survive repair radiation-induced damage, while some cells begin to express some of the radiation-induced nuclear and chomatin damage. Caffeine- and thymidine-treated, and untreated gamma-irradiated cell populations were studied with respect to the radiation-induced G2 delay, deficient DNA synthesis, and the appearance of cells with abnormal DNA contents. It is hypothesized that the measured deficiency in DNA synthesis observed in the first post-irradiation cell cycle may be a result of daughter cells from abnormal first post-irradiation mitoses

  20. Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

    Science.gov (United States)

    Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

    2009-09-25

    An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

  1. The cytometric future: it ain't necessarily flow!

    Science.gov (United States)

    Shapiro, Howard M

    2011-01-01

    Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.

  2. Serologic evidence of infection with granulocytic ehrlichiae in black bears in Pennsylvania.

    Science.gov (United States)

    Schultz, Sharon M; Nicholson, William L; Comer, James A; Childs, James E; Humphreys, Jan G

    2002-01-01

    Serum samples from 381 black bears (Ursus americanus) killed in Pennsylvania (USA) on 24 November 1997 were analyzed for antibodies reactive to the agent of human granulocytic ehrlichiosis (HGE; Ehrlichia sp.) by indirect immunofluorescence assay. Antibody reactivity to HGE antigen was detected in 21% (81/381) of the samples collected. Reactive samples were reported from 56% (14/25) of the counties where bear samples were collected. Endpoint antibody titer ranged from 1:8 to 1:16, 192, with a geometric mean titer of 1:582. There was no significant difference in antibody prevalence between male and female bears (P bears were significantly more likely to have reactive antibodies than juvenile bears (P bear blood clots (n = 181) through nested polymerase chain reaction assays were unsuccessful. Further studies are needed for identification of the pathogen-responsible for induction of HGE-reactive. This is the first description of antibodies reactive to the HGE agent in black bears and suggests these mammals are infected with the agent of HGE or an antigenically related ehrlichial species.

  3. Radioassay of granulocyte chemotaxis. Studies of human granulocytes and chemotactic factors. [/sup 51/Cr tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Gallin, J I

    1974-01-01

    The above studies demonstrate that the /sup 51/Cr radiolabel chemotactic assay is a relatively simple and objective means for studying leukocyte chemotaxis in both normal and pathological conditions. Application of this method to studies of normal human chemotaxis revealed a relatively narrow range of normal and little day-to-day variability. Analysis of this variability revealed that there is more variability among the response of different granulocytes to a constant chemotactic stimulus than among the chemotactic activity of different sera to a single cell source. Utilizing the /sup 51/Cr radioassay, the abnormal granulocyte chemotactic behavior reported in Chediak-Higashi syndrome and a patient with recurrent pyogenic infections and mucocutaneous candidiasis has been confirmed. The /sup 51/Cr chemotactic assay has also been used to assess the generation of chemotactic activity from human serum and plasma. The in vitro generation of two distinct chemotactic factors were examined; the complement product (C5a) and kallikrein, an enzyme of the kinin-generating pathway. Kinetic analysis of complement-related chemotactic factor formation, utilizing immune complexes or endotoxin to activate normal sera in the presence or absence of EGTA as well as kinetic analysis of activation of C2-deficient human serum, provided an easy means of distinguishing the classical (antibody-mediated) complement pathway from the alternate pathway. Such kinetic analysis is necessary to detect clinically important abnormalities since, after 60 min of generation time, normal chemotactic activity may be present despite complete absence or inhibition of one complement pathway. The chemotactic factor generated by either pathway of complement activation appears to be predominately attributable to C5a.

  4. Immunofluorescence detection of milk protein in meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2015-05-01

    Full Text Available Nowadays there are various vegetable protein additives intended for the manufacture of meat products in the food industry. These ingredients include both, plant-origin as well as animal-origin proteins. The most common vegetable additives include various types of flour, starch, fiber and plant protein. Among animal proteins, the most commonly used are plasma, collagen or milk protein. Milk protein is added to meat products due to its functional properties, such as emulsifying fats, improving the holding capacity of meat, improving juiciness, gel-forming capacity and affecting the taste of the product. Usage of these proteins, however, is currently limited by the effective legislation, not only in order to prevent consumer deception, but also because of their potential impact on consumers' health of. Thus, this issue has received considerable attention not only in the Czech Republic, but also globally. The main risk is the impossibility of selecting a suitable foodstuff for individuals with potential allergic reactions. The only option for allergic consumers to protect themselves is to strictly exclude the given allergen from their diet. Although the number of studies dealing with the reduction or loss of allergenicity is increasing, yet these practices are not common. Most of the population suffering from food allergies is thus still dependent on strict exclusion of foodstuffs causing adverse allergic reactions from their diet. Detection of allergens in foodstuffs is unfortunately quite difficult due to the fact that they occur in trace amounts and are often masked by different parts of the foodstuff. This research dealt with the detection of milk protein in meat products purchased in the market network of the Czech Republic, whereas declaration given by the manufacturer on the packaging for the small meat products purchased from the market was used to verify the detection of milk protein by the immunofluorescence method. 20 products were

  5. Immunofluorescent determination of wheat protein in meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2014-02-01

    foodstuffs is relatively difficult because of the fact that they occur in trace amounts and are often masked by various parts of the product. This paper deals with detection of wheat protein in meat products bought in the retail network of the Czech Republic. Ten cooked meat products, especially types of sausages and soft salami which stated wheat protein in their composition, were examined. The samples were processed using the method of immunofluorescence and stained with Texas Red fluorochrome. The presence of wheat protein was demonstrated in all the examined meat products. From the results it follows that the method of immunofluorescence is suitable for detection of wheat protein in meat products. Normal 0 21 false false false CS JA X-NONE

  6. Eighteen years experience of granulocyte donations-acceptable donor safety?

    Science.gov (United States)

    Axdorph Nygell, Ulla; Sollén-Nilsson, Agneta; Lundahl, Joachim

    2015-10-01

    Granulocyte transfusions are given to patients with life-threatening infections, refractory to treatment. The donors are stimulated with corticosteroids ± granulocyte colony stimulating factor (G-CSF). However, data regarding the donors' safety is sparse. The objective was therefore to evaluate short- and long-term adverse events (AE) in G-CSF stimulated donors. All consecutive granulocyte donors from 1994 to 2012 were identified through our registry. From the donation records, the number of aphereses, stimulation therapy, AE, blood values post donation, and recent status were evaluated. One hundred fifty-four volunteer donors were mobilized for 359 collections. Age at first granulocyte donation was 43 years (median; range 19-64 years). Follow-up was 60 months (median; range 0-229 months). The dose of G-CSF per collection was 3.8 ug/kg body weight (median; range 1.6-6.0 ug/kg). Sedimentation agent was HES. Short-term AE were mild. Blood values 4 weeks post donation with minor reductions/elevations mostly resolved in later donations. Fourteen donors were excluded from the registry due to hypertension (4), diabetes (2), atrial flutter (1), breast carcinoma (1), urethral carcinoma in situ (1), MGUS (1), thrombosis (1), anaphylaxis (1), primary biliary cirrhosis (1), and unknown (1). Three donors are deceased due to diabetes, acute myocardial infarction, and unknown cause. All excluded/deceased donors except one were excluded/died at least 6 months after first granulocyte donation. No serious short-term AE were observed. Due to the variability of diagnoses among excluded/deceased donors, we propose that it is less likely that granulocyte donations have a causative impact on these donors' exclusion or death. © 2014 Wiley Periodicals, Inc.

  7. Granulocytic sarcoma of the ovary in a nonleukemic patient.

    Science.gov (United States)

    Aguiar, R C; Pozzi, D H; Chamone, D A

    1993-01-01

    We report a case of granulocytic sarcoma of the ovary preceding acute myeloid leukemia by twelve months, with no evidence of any hematological involvement at the time of first diagnosis. The patient was initially treated with surgery and chemotherapy for undifferentiated lymphoma and, although this aggressive protocol resulted in a complete response, granulocytic sarcoma recurred as extramedullary disease, followed by the appearance of acute myeloid leukemia. We discuss the clinical, histopathological and immunohistochemical features of the disease, the differential diagnosis and, in particular, the role of early aggressive treatment on the outcome of the patient.

  8. IFDOTMETER: A New Software Application for Automated Immunofluorescence Analysis.

    Science.gov (United States)

    Rodríguez-Arribas, Mario; Pizarro-Estrella, Elisa; Gómez-Sánchez, Rubén; Yakhine-Diop, S M S; Gragera-Hidalgo, Antonio; Cristo, Alejandro; Bravo-San Pedro, Jose M; González-Polo, Rosa A; Fuentes, José M

    2016-04-01

    Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier. © 2015 Society for Laboratory Automation and Screening.

  9. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco

    2017-06-13

    Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

  10. Direct immunofluorescence of normal skin in rheumatoid arthritis.

    Science.gov (United States)

    Fitzgerald, O M; Barnes, L; Woods, R; McHugh, L; Barry, C; O'Loughlin, S

    1985-11-01

    The clinical significance of previously described immunoglobulin and complement deposition in the superficial dermal vessel walls of patients with rheumatoid arthritis is unknown. In the present study, skin biopsies were obtained from the normal forearm and buttock of 48 unselected patients with rheumatoid arthritis and were examined by direct immunofluorescence (IF) for the presence of immunoglobulin (IgG,A,M) and complement (C3) in the vessel walls. Deposits of C3, IgM or IgG were detected in 10 patients. Five patients had deposits at the forearm sample alone, four patients had deposits at both biopsy sites, while one patient was positive at the buttock alone. Clinical features were similar in patients with and without vessel IF. However, patients with IF were significantly more seropositive with lower levels of complement and raised levels of serum IgA and IgM. There was also an increased level of circulating IgG immune complexes in these patients. Further analysis following exclusion of seronegative patients revealed similar results. This study suggests that the presence of vessel IF identifies a subgroup of patients who have evidence of more severe immunological disturbance.

  11. Systemic lupus erythematosus and the Crithidia luciliae immunofluorescent test

    International Nuclear Information System (INIS)

    Whitehouse, I.J.; Fehr, K.; Wagenhaeuser, F.J.

    1983-01-01

    A comparative study of the Crithidia luciliae immunofluorescence (CL-IF) assay and an adapted Farr radioimmunoassay (RIA), for the measurement of antibodies to native deoxyribonucleic acid, was performed using forty-two sera from patients with systematic lupus erythematosus (SLE) and another forty-two from patients with rheumatoid arthritis. Both assays were specific for SLE. The CL-IF assay was statistically significantly more sensitive than the adapted RIA assay. This significant difference was due to greater sensitivity of the CL-IF assay in the cases of sera from patients with SLE of slight activity. Additional advantages of the CL-IF assay were its use to classify the immunoglobulin types of the antibodies (most commonly IgG or IgM) and to measure complement-fixing antibodies to native deoxyribonucleic acid; it affords a simple method of selecting and following SLE patients at risk of developing severe renal disease. These advantages plus the simplicity and inexpensiveness of the CL-IF assay make it a useful tool, especially for use in small laboratories, for the study of antibodies to native deoxyribonucleic acid in patients with SLE. (orig.) [de

  12. [Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

    Science.gov (United States)

    Storch, W

    1977-02-15

    By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

  13. Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent▿

    Science.gov (United States)

    Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

    2009-01-01

    Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 μg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 × 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 × g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health. PMID:19439525

  14. Undifferentiated granulocytic sarcoma: a case with epidural onset preceding acute promyelocytic leukemia.

    Science.gov (United States)

    Tosi, A; De Paoli, A; Fava, S; Luoni, M; Sironi, M; Tocci, A; Assi, A; Cassi, E

    1995-01-01

    This study reports a case of granulocytic sarcoma that developed in the epidural zone 25 days before clinical evidence of an acute promyelocytic leukemia. The case presented the diagnostic difficulties that are common to all aleukemic granulocytic sarcomas. Moreover, it highlights the very rare association between granulocytic sarcoma and acute promyelocytic leukemia, which is far from being explained.

  15. Imaging diagnosis of Granulocytic Sarcoma in the skull base

    International Nuclear Information System (INIS)

    Zheng Shaoyan; Xie Jiming; Yang Zhiyun; Zhou Zhou; Li Shurong

    2010-01-01

    Objective: To improve the understanding and imaging diagnosis of granulocytic sarcoma in the skull base. Methods: Three cases of granulocytic sarcomas in the skull base are reported. The clinical features and imaging findings were analyzed. Results: The three cases occurred in children with acute myeloid leukemia. Two patients presented with oculomotor paralysis before the diagnosis of leukemia, the third patient with history of leukemia presented with headache. Diffuse infiltration of basal skull bone marrow and extracranial soft tissue masses were shown on MRI. The signal intensities of the masses were similar to that of gray matter on T 1 WI and T 2 WI with marked contrast enhancement. The soft tissue masses were located in the para-sellar region and surrounded the lateral wall of the maxillary sinus in one case. The soft tissue mass of the second case infiltrated the orbital cavity, cavernous sinus and oculomotor nerve. Tumor infiltrating the meninges, cranial nerves and paranasal sinuses was seen in the third patient. Conclusion: Cranial nerve paralysis can be the presenting symptom of basal skull granulocytic sarcoma in children. Granulocytic sarcoma should be considered in the different diagnosis when diffuse abnormal signal intensities in the basal skull bone marrow with solitary or multiple soft tissue masses are shown on MRI. (authors)

  16. MRI findings of central nervous system granulocytic sarcoma (chloroma)

    International Nuclear Information System (INIS)

    Lee, Chang Man; Kim, Myung Soon; Kim, Ik Soo; Cho, Kwan Soo

    1997-01-01

    To characterize MRI findings of central nervous system (CNS) granulocytic sarcoma (chloroma) and to analyse the points which differentiate it from other CNS tumors. We evaluated MRI in six patients with CNS granulocytic sarcoma proven by surgery or bone marrow biopsy (intracranical, one case and spine five cases). A 0.5T superconductive MR machine was used for diagnosis and, axial, coronal and sagittal T1- and T2-weighted spin echo images and Gd-DTPA enhanced T1-weighted images were obtained. We retrospectively analized the location, signal intensity, margin, contrast enhancement and homogeneity, and bony change around the tumor. MRI findings of CNS granulocytic sarcomas were as follows : one tumor was seen to be an extra-axial mass in the posterior fossa of the brain, four were epidural, and one was an epidural and presacral masses in the spine;tumor magins were lobulated and three were smooth. On T1-weighted images, all tumors were of isoignal intensity;on T2-weighted images, four were of isosignal intersity and two were of high signal intensity. Contrast enhancement was inhomogeneous in five of six cases. Bony change around the tumor was seen in two cases. On T1-weighted images, CNS granulocytic sarcomas (chloromas) were of isosignal intensity, relative to brain parenchyma or spinal cord;on T2-weighted images, they were of iso or high signal intensity, with relative contrast enhancement. These points could be useful in differentiating them from other CNS tumors

  17. Indium-granulocyte scanning in the painful prosthetic joint

    International Nuclear Information System (INIS)

    Pring, D.J.; Henderson, R.G.; Keshavarzian, A.; Rivett, A.G.; Krausz, T.; Coombs, R.R.; Lavender, J.P.

    1986-01-01

    The value of indium-111-labeled granulocyte scanning to determine the presence of infection was assessed in 50 prosthetic joints (41 of which were painful) in 40 patients. Granulocytes were obtained from the patients' blood and labeled in plasma with indium 111 tropolonate. Abnormal accumulation of indium 111 in the region of the prosthesis was noted. Proven infection occurred in 11 prostheses, and all of the infections were detected by indium-111-labeled granulocyte scanning. Nineteen were not infected (including nine asymptomatic controls) and only two produced false-positive scans. This represents a specificity of 89.5%, sensitivity of 100%, and overall accuracy of 93.2%. These results compare favorably with plain radiography. There was no radiologic evidence of infection in three of the infected prostheses, and 10 of the noninfected prostheses had some radiologic features that suggested sepsis. We conclude that indium-granulocyte scanning can reliably detect or exclude infection in painful prosthetic joints and should prove useful in clinical management

  18. Granulocytes enzymes as a biomarker of radiotoxicity in exposed workers

    International Nuclear Information System (INIS)

    Milacic, S.; Jovicic, D.; Tanaskovic, I.; Marinkovic, O.; Milacic, S.)

    2007-01-01

    When radionuclide reaches the organism it causes internal irradiation and the lesions may be long lasting in various tissues. Enzymes in leukocytes will be used as a biomarkers of contamination with radio-nuclide in nuclear medicine workers. The analysed group had been consisted of 74 workers, exposed to radioactive isotopes J 131 and mTc 99 in nuclear medicine. Duration of occupational exposure (DOE) varied, so the groups with DOE of 1-5, 6-15, and 16-30 years, were compared to one another. The control group consisted of 52 subjects exposed to radionuclides (Cs 137 ) from environmental. Alkaline phosphatases and myeloperoxidase activity were inhibited in the granulocytes. The neutrophilic granulocytes count was lower while the number of eosinophils was higher

  19. Case report 432: Granulocytic sarcoma (GS), with hypereosinophilic syndrome (HES)

    Energy Technology Data Exchange (ETDEWEB)

    Xipell, J M; Beamish, M R; Clark, D

    1987-07-01

    A case is presented of a 28-year-old man with a history of the hypereosinophilic syndrome, who subsequently developed granulocytic sarcoma of the tibia which proved resistant to aggressive therapy. He ultimately developed acute myeloid leukemia which also was resistant to therapy. The disease process was characterized by localised skeletal pain, the development of lytic lesions in several areas of the skeleton and progression to frank myeloid leukemia. (orig./SHA).

  20. Sweet's Syndrome Successfully Treated with Granulocyte and Monocyte Adsorption Apheresis

    OpenAIRE

    Fujii, Asami; Mizutani, Yoko; Hattori, Yuki; Takahashi, Tomoko; Ohnishi, Hidenori; Yoshida, Shozo; Seishima, Mariko

    2017-01-01

    Sweet’s syndrome is a neutrophilic dermatosis characterized by an abrupt onset of painful erythematous lesions showing neutrophilic infiltrates in the dermis. Fever and an elevated neutrophil level are generally observed. Sweet’s syndrome may be idiopathic, malignancy-associated, or drug-induced (mainly involving granulocyte colony-stimulating factor (G-CSF) administration). Although systemic corticosteroids are usually effective, the symptoms of Sweet’s syndrome recur in some refractory case...

  1. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics.

    Directory of Open Access Journals (Sweden)

    Kornelis S M van der Geest

    Full Text Available Measuring changes of the T cell receptor (TCR repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

  2. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogona...

  3. Immunologic status of children with thyroid cancer living near Chernobyl (flow cytometric and electron microscopic study)

    International Nuclear Information System (INIS)

    Zak, K.P.; Gruzov, M.A.; Bolshova, B.V.; Afanasyeva, V.V.; Shlyakhovenko, V.S.; Vishnevskaya, O.A.; Tronko, N.D.

    1996-01-01

    It hag been carded out a light, election microscopic and flow cytometric study of blood leukocyte of children with malignant tumors (papillary carcinoma) of thyroid gland who were living at the moment of the accident near Chernobyl. The results obtained point out the presence of some disturbances of immune status of these children

  4. Cluster Analysis of Flow Cytometric List Mode Data on a Personal Computer

    NARCIS (Netherlands)

    Bakker Schut, Tom C.; Bakker schut, T.C.; de Grooth, B.G.; Greve, Jan

    1993-01-01

    A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce

  5. A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

    DEFF Research Database (Denmark)

    Holm, C.; Mathiasen, T.; Jespersen, Lene

    2004-01-01

    were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine...

  6. Neutrophilic granulocytes reactive response in candida vulvovaginitis patients with intracellular microorganism persistence complications

    OpenAIRE

    YAKOVYCHUK NINA DMYTRIVNA; DJUIRIAK VALENTYNA STEPANIVNA

    2015-01-01

    Polymorphic neutrophilic granulocytes reactive response and body immune reactivity in general considerably decrease in patients suffering from candida vaginitis on the basis of intracellular microorganisms persistence.

  7. Characterization of buffy coat-derived granulocytes for clinical use: a comparison with granulocyte colony-stimulating factor/dexamethasone-pretreated donor-derived products.

    Science.gov (United States)

    van de Geer, A; Gazendam, R P; Tool, A T J; van Hamme, J L; de Korte, D; van den Berg, T K; Zeerleder, S S; Kuijpers, T W

    2017-02-01

    Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte colony-stimulating factor (G-CSF). The latter is - dependent on the local and national settings - obtained following a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. We pooled ten buffy coats following the production of platelet concentrates. Saline 0·9% was added to decrease the viscosity and the product was split into plasma, red cells and a 'super' buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in vitro killing of Staphylococcus aureus, Escherichia coli and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal; especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily available in the regular blood production process and would take away the concerns around the apheresis product. The product described appears a promising alternative for transfusion purposes. © 2017 International Society of Blood Transfusion.

  8. Congenital hypogammaglobulinemia associated with granulocyte disorders. A case presentation

    International Nuclear Information System (INIS)

    Sanchez Segura, Miriam C; Marsan Suarez, Vianed; Socarras Ferrer, Bertha B; Ojeda de Leon, Norma

    2009-01-01

    This is the case of a child aged 11 months with a history of systemic sepsis from Pseudomona aeruginosa at 5 months, neutropenia, leucopenia, sepsis-associated anemia and from then, recurrent acute respiratory infections of the high respiratory tract, allergic manifestations and furunculosis from pseudomona. Immunologic study conducted showed a decreased figure of IgG with a light increase of CD4 +c ooperative-IgM of T cells. Also, we found the presence of neutropenia and marked defect of phagocytosis. We made the diagnosis of granulocyte-associate congenital hypogammaglobulinemia. The patient was treated with human gamma globulin by intramuscular route, transference factor and immunoferon, with an obvious improvement

  9. Positive /sup 111/In-granulocyte scintigraphy in a patient with focal leukemic blast cell infiltrations

    Energy Technology Data Exchange (ETDEWEB)

    Syrjaelae, M; Remes, K; Paavonen, T; Liewendahl, K

    1985-06-01

    A patient with acute myeloid leukemia was investigated with /sup 111/In-granulocyte scintigraphy to reveal possible sites of infection. /sup 111/In-granulocytes accumulated in areas of leukemia blast cell infiltration leading to a false-positive scintigram. This possibility must be kept in mind when studying leukemic patients using labeled leukocytes.

  10. 99Tcsup(m)-HMPAO labelled leucocytes: comparison with 111In-tropolonate labelled granulocytes

    International Nuclear Information System (INIS)

    Peters, A.M.; Roddie, M.E.; Zacharopoulos, G.P.; George, P.; Stuttle, A.W.J.; Lavender, J.P.; Danpure, H.J.; Osman, S.

    1988-01-01

    The lipophilic complex, 99 Tcsup(m)-hexamethylpropyleneamine oxime (HMPAO) is an efficient leucocyte label, and labels granulocytes with more stability than mononuclear leucocytes. The recovery of 99 Tcsup(m)-HMPAO granulocytes was similar to 111 In-labelled granulocytes isolated and labelled in plasma using tropolone. The Tsub(1/2) of 99 Tcsup(m)-HMPAO labelled granulocytes in blood was less than that of 111 In-labelled granulocytes. The initial biodistribution of 99 Tcsup(m)-labelled leucocytes was similar to 111 In-labelled granulocytes, with a rapid initial lung transit, prominent splenic activity, bone marrow activity and minimal hepatic activity, although, unlike 111 In, 99 Tcsup(m) activity was also seen in urine, occasionally in the gallbladder, and, from about 4 h, consistently in the colon. Bone marrow activity was particularly prominent with 99 Tcsup(m). About 6% of 99 Tcsup(m) was excreted in the faeces up to 48 h after injection, and about 17% in urine up to 24 h. The time-activity curves of reticuloendothelial activity up to 24 h were broadly similar for the two labelled cell preparations. Clinical information given by the two agents was similar in 27 of 30 patients who received both. We conclude that with respect to granulocyte kinetics and clinical data, 99 Tcsup(m)-HMPAO labelled leucocytes are comparable with 111 In-tropolonate labelled granulocytes. (author)

  11. Image cytometric nuclear texture features in inoperable head and neck cancer: a pilot study

    International Nuclear Information System (INIS)

    Strojan-Flezar, Margareta; Lavrencak, Jaka; Zganec, Mario; Strojan, Primoz

    2011-01-01

    Image cytometry can measure numerous nuclear features which could be considered a surrogate end-point marker of molecular genetic changes in a nucleus. The aim of the study was to analyze image cytometric nuclear features in paired samples of primary tumor and neck metastasis in patients with inoperable carcinoma of the head and neck. Image cytometric analysis of cell suspensions prepared from primary tumor tissue and fine needle aspiration biopsy cell samples of neck metastases from 21 patients treated with concomitant radiochemotherapy was performed. Nuclear features were correlated with clinical characteristics and response to therapy. Manifestation of distant metastases and new primaries was associated (p<0.05) with several chromatin characteristics from primary tumor cells, whereas the origin of index cancer and disease response in the neck was related to those in the cells from metastases. Many nuclear features of primary tumors and metastases correlated with the TNM stage. A specific pattern of correlation between well-established prognostic indicators and nuclear features of samples from primary tumors and those from neck metastases was observed. Image cytometric nuclear features represent a promising candidate marker for recognition of biologically different tumor subgroups

  12. Granulocytes in Helminth Infection - Who is Calling the Shots?

    Science.gov (United States)

    Makepeace, BL; Martin, C; Turner, JD; Specht, S

    2012-01-01

    Helminths are parasitic organisms that can be broadly described as “worms” due to their elongated body plan, but which otherwise differ in shape, development, migratory routes and the predilection site of the adults and larvae. They are divided into three major groups: trematodes (flukes), which are leaf-shaped, hermaphroditic (except for blood flukes) flatworms with oral and ventral suckers; cestodes (tapeworms), which are segmented, hermaphroditic flatworms that inhabit the intestinal lumen; and nematodes (roundworms), which are dioecious, cylindrical parasites that inhabit intestinal and peripheral tissue sites. Helminths exhibit a sublime co-evolution with the host´s immune system that has enabled them to successfully colonize almost all multicellular species present in every geographical environment, including over two billion humans. In the face of this challenge, the host immune system has evolved to strike a delicate balance between attempts to neutralize the infectious assault versus limitation of damage to host tissues. Among the most important cell types during helminthic invasion are granulocytes: eosinophils, neutrophils and basophils. Depending on the specific context, these leukocytes may have pivotal roles in host protection, immunopathology, or facilitation of helminth establishment. This review provides an overview of the function of granulocytes in helminthic infections. PMID:22360486

  13. A karyometric note on nucleoli in human early granulocytic precursors.

    Science.gov (United States)

    Smetana, K; Mikulenková, D; Jirásková, I; Klamová, H

    2006-01-01

    The diameter of nucleoli was measured in human bone marrow early granulocytic precursors after visualization by a simple cytochemical method for demonstration of RNA. Such method facilitated to clearly see nucleolar bodies without perinucleolar chromatin, including those of micronucleoli. The bone marrow of patients suffering from chronic myeloid leukaemia (untreated with cytostatics) provided a satisfactory number of both myeloblasts and promyelocytes for nucleolar measurements because of prevailing granulopoiesis. The direct nucleolar measurement was carried out on digitized and processed images on the screen at magnification 4,300x. It seems to be likely that the nucleolar size is directly related to the number of nucleoli per cell. The largest nucleoli were present in both myeloblasts and promyelocytes that possessed a single nucleolus. In contrast, the nucleolar diameter was significantly smaller in cells with multiple nucleoli. However, in cells with small multiple nucleoli, one of them was always larger and dominant with a large number of AgNORs. Such large nucleoli are possibly visible in specimens stained with panoptic procedures or methods staining nuclear chromatin or DNA. It should also be mentioned that both myeloblasts and promyelocytes mostly possessed two nucleoli with the mean diameter close to 1.5 microm. The incidence of early granulocytic precursors classified according to the nucleolar number and size strongly suggested that the various nucleolar number and nucleolar size in these cells might be related to the different stage of the cell cycle and might also explain their heterogeneity.

  14. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.

    2008-01-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  15. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling

    1990-01-01

    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge...

  16. Serologic Evidence of Human Monocytic and Granulocytic Ehrlichiosis in Israel

    Science.gov (United States)

    Keysary, Avi; Amram, Lili; Keren, Gershon; Sthoeger, Zev; Potasman, Israel; Jacob, Amir; Strenger, Carmella; Dawson, Jacqueline E.

    1999-01-01

    We conducted a retrospective serosurvey of 1,000 persons in Israel who had fever of undetermined cause to look for Ehrlichia chaffeensis antibodies. Four of five cases with antibodies reactive to E. chaffeensis were diagnosed in the summer, when ticks are more active. All patients had influenzalike symptoms with high fever. None of the cases was fatal. Three serum samples were also seroreactive for antibodies to E. canis, and one was also reactive to the human granulocytic ehrlichiosis (HGE) agent. The titer to the HGE agent in this patient was higher than the serum titer to E. chaffeensis, and the Western blot analysis also indicated that the HGE agent was the primary cause of infection. We present the first serologic evidence that the agents of human monocytic ehrlichiosis (HME) and HGE are present in Israel. Therefore, human ehrlichiosis should be included in the differential diagnoses for persons in Israel who have been exposed to ticks and have influenzalike symptoms. PMID:10603210

  17. Generation of dendritic cells for immunotherapy is minimally impaired by granulocytes in the monocyte preparation.

    Science.gov (United States)

    ten Brinke, Anja; Karsten, Miriam L; Dieker, Miranda C; Zwaginga, Jaap Jan; Vrielink, Hans; Marieke van Ham, S

    2006-01-01

    The growing number of clinical studies, using monocyte-derived DC therapy, requires protocols where a sufficient number of dendritic cell (DCs) are produced according to current Good Manufacturing Practice guidelines. Therefore, a closed culture system for the generation of DCs is inevitable. One cost-effective way to isolate monocytes directly from leukapheresis material in a closed system is by elutriation with the Elutra cell separation system. In the Elutra, granulocytes co-purify with the monocytes. Therefore, we studied if and to what extent the presence of granulocytes in a monocyte product affects the generation of mature DCs. The presence of up to 16% granulocytes in the monocyte product had no significant effects on the quality of the DCs formed. The presence of higher granulocyte percentages, however, gradually altered DC quality. In this respect, the presence of higher number of granulocytes induced significant lower migratory capacity of the DCs and lower expression levels of CD80, CD40 and CD86. No effects were observed on the DC yield, cytokine production or the stimulatory capacity of the DCs in MLR. In conclusion, the presence of 20-30% granulocytes in a monocyte product has no major influence on the quality of the DCs generated from monocytes. Therefore, the Elutra is a suitable closed system apparatus to separate monocytes from other blood components for the generation of DCs, even from leukapheresis material which contains a high number of granulocytes.

  18. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Science.gov (United States)

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

    2014-05-01

    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    Science.gov (United States)

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  20. Pre leukemic granulocytic sarcoma of vagina: a case report with review of literature

    International Nuclear Information System (INIS)

    Lakshminarasimhan, Srinivasan; Doval, D.C.; Rajashekhar, Usha; Mukherjee, Geethashree; Kannan, V.; Lakshmi Devi; Bapsy, P.P.

    1996-01-01

    Granulocytic sarcoma is an extramedullary tumor of malignant granulocytic progenitor cells, that may precede the onset of acute myeloid leukemia or appear during the leukemic manifestation or blastic crisis of chronic myeloproliferative disorders. A case of granulocytic sarcoma of vagina in a 27 year old woman treated with local radiotherapy is described. After seven months of follow up she developed acute myeloid leukemia. The case has been presented in view of its rarity and discussed in light of the available literature. (author). 13 refs., 1 fig

  1. Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura

    Directory of Open Access Journals (Sweden)

    D Cavallo

    2010-01-01

    Full Text Available Karyological and flow cytometric (FCM analyses were performed on a group of 14 green toads of the Bufo viridis species from seven Eurasian populations. Both approaches gave concordant results concerning the DNA ploidy level. All the populations examined were represented exclusively by diploid or tetraploid specimens, except one, where triploids were found. Results evidenced an interpopulation variability in DNA content against the same ploidy level, as well as an unusually high number of triploids in a particular reproductive place. The origin of polyploidy and the presence and persistence of a high number of triploids in a particular population are discussed.

  2. Use of LysoTracker dyes: a flow cytometric study of autophagy.

    Science.gov (United States)

    Chikte, Shaheen; Panchal, Neelam; Warnes, Gary

    2014-02-01

    The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process and to compare this with the upregulation of autophagy marker, the microtubule-associated protein LC3B. Although the mechanism of action of LysoTracker dyes is not fully understood, they have been used in microscopy to image acidic spherical organelles, and their use in flow cytometry has not been thoroughly investigated in the study of autophagy. This investigation uses numerous autophagy-inducing agents including chloroquine (CQ), rapamycin, low serum (used to analyze patient cells as well as easier to use and significantly less costly. Copyright © 2013 International Society for Advancement of Cytometry.

  3. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...

  4. Flow cytometric of reticulocytes quantification: radio-induction medullary aplasia application

    International Nuclear Information System (INIS)

    Dubner, D.; Perez, M.; Gisone, P.

    1996-01-01

    Flow cytometric reticulocyte quantification was assayed in ten patients undergoing bone marrow transplantation (BMT) with previous conditioning by chemotherapy and total body irradiation. A reticulocyte maturity index (RMI) was determined taking into account the RNA content. With the aim of testing the utility of RMI as an early predictor of functional recovery in marrow aplasia, other hematological indicators as neutrophils count were comparatively evaluated. Mean time elapsed between BMT and engraftment evidence by RMI was 17,6 days. In six patients the RMI was the earliest indicator of functional recovery. The applicability of this assay in the pursuit of radioinduced bone marrow aplasia is discussed. (authors). 4 refs., 4 figs., 2 tabs

  5. Granulocytic sarcoma in a patient with chronic myeloid leukaemia in complete haematological, cytogenetic and molecular remission.

    Science.gov (United States)

    Kittai, Adam; Yu, Eun-Mi; Tabbara, Imad

    2014-12-23

    Granulocytic sarcoma, also known as myeloid sarcoma, is an extramedullary tumour composed of immature myeloid cells. Granulocytic sarcoma is typically found in patients with acute myeloid leukaemia, accelerated phase or blast crisis of chronic myeloid leukaemia, myelodysplastic syndrome, or as an isolated event without bone marrow involvement. We present a case of granulocytic sarcoma in a patient with chronic myeloid leukaemia in the setting of complete haematological, molecular and cytogenetic remission. Our patient was first treated with imatinib for chronic-phase chronic myeloid leukaemia. After maintaining remission for 42 months, he developed a granulocytic sarcoma in his spine. In this case report, we describe our case, along with the three other cases reported in the literature. In addition to being a rare diagnosis, this case demonstrates the importance of being vigilant in diagnosing the cause of back pain and atypical symptoms in patients with a history of leukaemia. 2014 BMJ Publishing Group Ltd.

  6. Modulation of oxygen-dependent and oxygen-independent metabolism of neutrophilic granulocytes by quantum points.

    Science.gov (United States)

    Pleskova, S N; Mikheeva, E R

    2011-08-01

    Inhibition of neutrophilic granulocyte metabolism under the effect of semiconductor quantum points was demonstrated. The status of the oxidative system was evaluated by the NBT test, nonoxidative status by the lysosomal cationic test. It was found that quantum points in a dose of 0.1 mg/ml irrespective of their core and composition of coating significantly inhibited oxygen-dependent and oxygen-independent metabolism of neutrophilic granulocytes.

  7. Granulocytic Sarcoma in a Nonleukemic Patient: Place of Radiotherapy and Systemic Therapies

    Directory of Open Access Journals (Sweden)

    C. Chargari

    2011-01-01

    Full Text Available Granulocytic sarcoma is a rare extramedullary tumour, which most often occurs in the course of an acute or chronic leukaemia or myeloproliferative disorders. Rarely it is found before peripheral blood or bone marrow evidence of leukemia is present. We report an unusual case of acute paraplegia at first presentation of a spinal epidural granulocytic sarcoma without any haematological disorder. Therapeutic strategies are discussed in the light of the literature.

  8. Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

    Science.gov (United States)

    Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

    2012-12-01

    The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. © 2011 Blackwell Publishing Ltd.

  9. Quality control in the application of flow cytometric assays of genetic damage due to environmental contaminants

    International Nuclear Information System (INIS)

    McCreedy, C.D.; Jagoe, C.H.; Brisbin, I.L. Jr.; Wentworth, R.W.; Dallas, C.E.

    1995-01-01

    Clinical technologies, such as flow cytometry, are increasingly adopted by environmental toxicologists to identify resource damage associated with exposure to xenobiotics. One application of flow cytometry allows the rapid determination of the DNA content of large numbers of individual cells, and can be used to detect aneuploidy or other genetic abnormalities. The laboratory has used this methodology in studies of genetic toxicology of fish, birds, arid mammals exposed to organic pollutants, metals and radionuclides, However, without appropriate quality controls, false positive results and other artifacts can arise from sample handling and preparations, inter and intra-individual variations, instrument noise and other sources. The authors describe the routine measures this laboratory employs to maintain quality control of genomic DNA analysis, including the control of staining conditions, machine standardization, pulse-width doublet discrimination, and, in particular, the use of internal controls and the use of time as a cytometric parameter. Neglect of these controls can produce erroneous results, leading to conclusions of genetic abnormalities when none are present. Conversely, attention to these controls, routinely used in clinical settings, facilitates the interpretation of flow cytometric data and allows the application of this sensitive indicator of genotoxic effects to a variety of environmental problems

  10. Vortex-dislodged cells from bone marrow trephine biopsy yield satisfactory results for flow cytometric immunophenotyping.

    Science.gov (United States)

    Bommannan, K; Sachdeva, M U S; Gupta, M; Bose, P; Kumar, N; Sharma, P; Naseem, S; Ahluwalia, J; Das, R; Varma, N

    2016-10-01

    A good bone marrow (BM) sample is essential in evaluating many hematologic disorders. An unsuccessful BM aspiration (BMA) procedure precludes a successful flow cytometric immunophenotyping (FCI) in most hematologic malignancies. Apart from FCI, most ancillary diagnostic techniques in hematology are less informative. We describe the feasibility of FCI in vortex-dislodged cell preparation obtained from unfixed trephine biopsy (TB) specimens. In pancytopenic patients and dry tap cases, routine diagnostic BMA and TB samples were complemented by additional trephine biopsies. These supplementary cores were immediately transferred into sterile tubes filled with phosphate-buffered saline, vortexed, and centrifuged. The cell pellet obtained was used for flow cytometric immunophenotyping. Of 7955 BMAs performed in 42 months, 34 dry tap cases were eligible for the study. Vortexing rendered a cell pellet in 94% of the cases (32 of 34), and FCI rendered a rapid diagnosis in 100% of the cases (32 of 32) where cell pellets were available. We describe an efficient procedure which could be effectively utilized in resource-limited centers and reduce the frequency of repeat BMA procedures. © 2016 John Wiley & Sons Ltd.

  11. Flow cytometric analysis of microbial contamination in food industry technological lines--initial study.

    Science.gov (United States)

    Józwa, Wojciech; Czaczyk, Katarzyna

    2012-04-02

    Flow cytometry constitutes an alternative for traditional methods of microorganisms identification and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer) may be analysed directly. This leads to a significant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the flow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with flow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and labor-saving detection of microbial contamination in food industry.

  12. A flow cytometric assay technology based on quantum dots-encoded beads

    International Nuclear Information System (INIS)

    Wang Haiqiao; Liu Tiancai; Cao Yuancheng; Huang Zhenli; Wang Jianhao; Li Xiuqing; Zhao Yuandi

    2006-01-01

    A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

  13. Image Cytometric Analysis of Algal Spores for Evaluation of Antifouling Activities of Biocidal Agents.

    Science.gov (United States)

    Il Koo, Bon; Lee, Yun-Soo; Seo, Mintae; Seok Choi, Hyung; Leng Seah, Geok; Nam, Taegu; Nam, Yoon Sung

    2017-07-31

    Chemical biocides have been widely used as marine antifouling agents, but their environmental toxicity impose regulatory restriction on their use. Although various surrogate antifouling biocides have been introduced, their comparative effectiveness has not been well investigated partly due to the difficulty of quantitative evaluation of their antifouling activity. Here we report an image cytometric method to quantitatively analyze the antifouling activities of seven commercial biocides using Ulva prolifera as a target organism, which is known to be a dominant marine species causing soft fouling. The number of spores settled on a substrate is determined through image analysis using the intrinsic fluorescence of chlorophylls in the spores. Pre-determined sets of size and shape of spores allow for the precise determination of the number of settled spores. The effects of biocide concentration and combination of different biocides on the spore settlement are examined. No significant morphological changes of Ulva spores are observed, but the amount of adhesive pad materials is appreciably decreased in the presence of biocides. It is revealed that the growth rate of Ulva is not directly correlated with the antifouling activities against the settlement of Ulva spores. This work suggests that image cytometric analysis is a very convenient, fast-processable method to directly analyze the antifouling effects of biocides and coating materials.

  14. Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes

    International Nuclear Information System (INIS)

    Butterick, C.J.; Baehner, R.L.; Boxer, L.A.; Jersild, R.A. Jr.

    1983-01-01

    The cellular sites of H 2 O 2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1- 14 C oxidation to 14 CO 2 . Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H 2 O 2 in the phagocytizing granulocyte

  15. Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

    Science.gov (United States)

    Forsey, T; Darougar, S

    1980-02-01

    A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

  16. Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

    Science.gov (United States)

    Zapata, M; Chernesky, M; Mahony, J

    1984-06-01

    Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.

  17. Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

    OpenAIRE

    Zapata, M; Chernesky, M; Mahony, J

    1984-01-01

    Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.

  18. Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

    Science.gov (United States)

    Schmitz, H

    1981-01-01

    An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

  19. Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

    Science.gov (United States)

    Shirol, Pallavi D; Naik, Veena; Kale, Alka

    2015-10-01

    Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

  20. Tissue-transglutaminase contributes to neutrophil granulocyte differentiation and functions.

    Science.gov (United States)

    Balajthy, Zoltán; Csomós, Krisztián; Vámosi, György; Szántó, Attila; Lanotte, Michel; Fésüs, László

    2006-09-15

    Promyelocytic NB4 leukemia cells undergo differentiation to granulocytes following retinoic acid treatment. Here we report that tissue transglutaminase (TG2), a protein cross-linking enzyme, was induced, then partially translocated into the nucleus, and became strongly associated with the chromatin during the differentiation process. The transglutaminase-catalyzed cross-link content of both the cytosolic and the nuclear protein fractions increased while NB4 cells underwent cellular maturation. Inhibition of cross-linking activity of TG2 by monodansylcadaverin in these cells led to diminished nitroblue tetrazolium (NBT) positivity, production of less superoxide anion, and decreased expression of GP91PHOX, the membrane-associated subunit of NADPH oxidase. Neutrophils isolated from TG2(-/-) mice showed diminished NBT reduction capacity, reduced superoxide anion formation, and down-regulation of the gp91phox subunit of NADPH oxidase, compared with wild-type cells. It was also observed that TG2(-/-) mice exhibited increased neutrophil phagocytic activity, but had attenuated neutrophil chemotaxis and impaired neutrophil extravasation with higher neutrophil counts in their circulation during yeast extract-induced peritonitis. These results clearly suggest that TG2 may modulate the expression of genes related to neutrophil functions and is involved in several intracellular and extracellular functions of extravasating neutrophil.

  1. Granulocyte-associated IgG in neutropenic disorders

    International Nuclear Information System (INIS)

    Cines, D.B.; Passero, F.; Guerry, D.; Bina, M.; Dusak, B.; Schreiber, A.D.

    1982-01-01

    We applied a radiolabeled antiglobulin test to a study of patients with a variety of neutropenic disorders. After defining the nature of the interaction of radiolabeled anti-IgG with the neutrophil, we studied 16 patients with neutropenia of uncertain etiology and adequate bone marrow granulocyte precursors. Twelve of these 16 patients had increased neutrophil-associated IgG (PMN-IgG). Patients with the highest levels of PMN-IgG had the lowest neutrophil counts. The majority of patients with neutropenia and increased PMN-IgG had an underlying immunologic disorder that included immune thrombocytopenic purpura in 5 patients and autoimmune hemolytic anemia in 1 patient. In some patients, elevated PMN-IgG preceded other evidence for immunologic disease. The direct antiglobulin test helped to distinguish neutropenic patients with increased PMN-IgG both from patients with neutropenia due to a known nonimmune disorder and from nonneutropenic patients with rheumatoid arthritis or systemic lupus erythematosis. Each of four patients with increased neutrophil-associated IgG treated with systemic corticosteroids responded clinically with an associated fall in neutrophil IgG and a rise in the circulating neutrophil count. The radiolabeled antiglobulin test appears useful in defining a subpopulation of patients with neutropenia due to an underlying immunologic disorder

  2. Granulocyte colony-stimulating factor induces in vitro lymphangiogenesis

    International Nuclear Information System (INIS)

    Lee, Ae Sin; Kim, Dal; Wagle, Susbin Raj; Lee, Jung Eun; Jung, Yu Jin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

    2013-01-01

    Highlights: •G-CSF induces tube formation, migration and proliferation of lymphatic cells. •G-CSF increases phosphorylation of MAPK and Akt in lymphatic endothelial cells. •MAPK and Akt pathways are linked to G-CSF-induced in vitro lymphangiogenesis. •G-CSF increases sprouting of a lymphatic ring. •G-CSF produces peritoneal lymphangiogenesis. -- Abstract: Granulocyte-colony stimulating factor (G-CSF) is reported to induce differentiation in cells of the monocyte lineage and angiogenesis in vascular endothelial cells, but its effects on lymphangiogenesis is uncertain. Here we examined the effects and the mechanisms of G-CSF-induced lymphangiogenesis using human lymphatic endothelial cells (hLECs). Our results showed that G-CSF induced capillary-like tube formation, migration and proliferation of hLECs in a dose- and time-dependent manner and enhanced sprouting of thoracic duct. G-CSF increased phosphorylation of Akt and ERK1/2 in hLECs. Supporting the observations, specific inhibitors of phosphatidylinositol 3′-kinase and MAPK suppressed the G-CSF-induced in vitro lymphangiogenesis and sprouting. Intraperitoneal administration of G-CSF to mice also stimulated peritoneal lymphangiogenesis. These findings suggest that G-CSF is a lymphangiogenic factor

  3. Exercise increases lactoferrin, but decreases lysozyme in salivary granulocytes.

    Science.gov (United States)

    Gillum, Trevor; Kuennen, Matthew; McKenna, Zachary; Castillo, Micaela; Jordan-Patterson, Alex; Bohnert, Caitlin

    2017-05-01

    Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise. To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45 + CD15 + cells. 11 males (20.3 ± 0.8 years, 57.2 ± 7.6 mL/kg/min V̇O 2pk , 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO 2pk . 12 mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45 + ) and granulocytes (CD45 + CD15 + ) using flow cytometry. Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

  4. Red cell ferritin and iron stores in chronic granulocytic leukemia

    International Nuclear Information System (INIS)

    Cermak, J.; Neuwirth, J.; Voglova, J.; Brabec, V.; Chrobak, L.

    1994-01-01

    Basic red cell ferritin was investigated in 28 patients with different phases of chronic granulocytic leukemia (GCL). Red cell ferritin was significantly decreased in remission after busulphan treatment and significantly elevated in the blast crisis as compared to healthy controls. Bone marrow stainable iron was decreased or absent in 86% of patients in the initial phase at the time of diagnosis and in 92% of those in remission. Red cell ferritin correlated with serum ferritin, however, serum ferritin level remained above normal range during all phases of the disease. A negative correlation between red cell ferritin and hemoglobin (Hb) (r = -0.605, p < 0.001) suggested that red cell ferritin level reflected the rate of iron utilization for heme synthesis. Decrease red cell iron observed in the remission may be explained by regression of dyserythropoiesis and by restoration of normal Hb synthesis after busulphan treatment. A progressive dyserythropoiesis in the blast crisis may lead to an increased red cell ferritin level. (author)

  5. Complement activated granulocytes can cause autologous tissue destruction in man

    Directory of Open Access Journals (Sweden)

    E. Löhde

    1992-01-01

    Full Text Available Activation of polymorphonuclear granulocytes (PMNs by C5a is thought to be important in the pathogenesis of multiple organ failure during sepsis and after trauma. In our experiment exposure of human PMNs to autologous zymosan activated plasma (ZAP leads to a rapid increase in chemiluminescence. Heating the ZAP at 56°C for 30 min did not alter the changes, while untreated plasma induced only baseline activity. The respiratory burst could be completely abolished by decomplementation and preincubation with rabbit antihuman C5a antibodies. Observation of human omentum using electron microscopy showed intravascular aggregation of PMNs, with capillary thrombosis and diapedesis of the cells through endothelial junctions 90 s after exposure to ZAP. PMNs caused disruption of connections between the mesothelial cells. After 4 min the mesothelium was completely destroyed, and connective tissue and fat cells exposed. Native plasma and minimum essential medium did not induce any morphological changes. These data support the concept that C5a activated PMNs can cause endothelial and mesothelial damage in man. Even though a causal relationship between anaphylatoxins and organ failure cannot be proved by these experiments C5a seems to be an important mediator in the pathogenesis of changes induced by severe sepsis and trauma in man.

  6. Utility of Immature Granulocyte Percentage in Pediatric Appendicitis

    Science.gov (United States)

    Mathews, Eleanor K.; Griffin, Russell L.; Mortellaro, Vincent; Beierle, Elizabeth A.; Harmon, Carroll M.; Chen, Mike K.; Russell, Robert T.

    2014-01-01

    Background Acute appendicitis is the most common cause of abdominal surgery in children. Adjuncts are utilized to help clinicians predict acute or perforated appendicitis, which may affect treatment decisions. Automated hematologic analyzers can perform more accurate automated differentials including immature granulocyte percentages (IG%). Elevated IG% has demonstrated improved accuracy for predicting sepsis in the neonatal population than traditional immature to total neutrophil count (I/T) ratios. We intended to assess the additional discriminatory ability of IG% to traditionally assessed parameters in the differentiation between acute and perforated appendicitis. Materials and Methods We identified all patients with appendicitis from July 2012 to June 2013 by ICD-9 code. Charts were reviewed for relevant demographic, clinical, and outcome data, which were compared between acute and perforated appendicitis groups using Fischer’s exact and t-test for categorical and continuous variables, respectively. We utilized an adjusted logistic regression model utilizing clinical lab values to predict the odds of perforated appendicitis. Results 251 patients were included in the analysis. Those with perforated appendicitis had a higher white blood cell (WBC) count (p=0.0063), C-reactive protein (CRP) (pappendicitis. The c-statistic of the final model was 0.70, suggesting fair discriminatory ability in predicting perforated appendicitis. Conclusions IG% did not provide any additional benefit to elevated CRP and presence of left shift in the differentiation between acute and perforated appendicitis. PMID:24793450

  7. Paclitaxel, ifosfamide and cisplatin with granulocyte colony-stimulating factor or recombinant human interleukin 3 and granulocyte colony-stimulating factor in ovarian cancer : A feasibility study

    NARCIS (Netherlands)

    Veldhuis, GJ; Willemse, PHB; Beijnen, JH; Piersma, H; vanderGraaf, WTA; deVries, EGE; Boonstra, J.

    1997-01-01

    The tolerability and efficacy of four courses of paclitaxel and ifosfamide plus cisplatin every 3 weeks was evaluated in patients with residual or refractory ovarian cancer. Additionally, supportive haematological effects of recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte

  8. Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

    International Nuclear Information System (INIS)

    Yu, Hye-Weon; Kim, In S.; Niessner, Reinhard; Knopp, Dietmar

    2012-01-01

    Highlights: ► First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. ► Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. ► The two model target analytes were low molecular weight compounds of microbial and chemical origin. ► The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC 50 values of 5 μg L −1 and 1.1 μg L −1 and dynamic ranges of 0.52–30 μg L −1 and 0.13–10 μg L −1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled

  9. Increased Granulocyte Heparanase Activity in Neutrophils from Patients with Lupus Nephritis and Idiopathic Membranous Nephropathy.

    Science.gov (United States)

    Szymczak, Maciej; Kuźniar, Jakub; Kopeć, Wacław; Żabińska, Marcelina; Marchewka, Zofia; Kościelska-Kasprzak, Katarzyna; Klinger, Marian

    2017-02-01

    Heparanase is a β-glucuronidase that cleaves sugar chains of heparan sulfate proteoglycans. It is believed that heparanase may be involved in the pathogenesis of proteinuria. The aim of this study was to assess the significance of heparanase in the pathogenesis of particular glomerulonephritis types. The evaluation of heparanase activity in serum, urine, and granulocytes and superoxide dismutase (SOD) activity in granulocytes of patients with lupus nephritis (n = 17), membranous nephropathy (n = 11), IgA nephropathy (n = 12), focal and segmental glomerulosclerosis (n = 18), mesangiocapillary glomerulonephritis (n = 12) and in 19 healthy volunteers were performed. The heparanase activity in granulocytes of patients with lupus nephritis and membranous nephropathy was higher than heparanase activity in granulocytes in the control group (p = 0.02 in both cases). This is the first observation of this phenomenon. There was no difference between SOD activity in granulocytes of patients with all assessed types of glomerulonephritis and the control group. A positive correlation between heparanase activity in urine and double-strain DNA antibodies (r = 0.51; p = 0.04), and reverse correlations between heparanase in urine and hemolytic activity of the complement (r = -0.57; p = 0.03) in the lupus nephritis group, and between heparanase activity in granulocytes and serum total protein level (r = -0.69; p = 0.02) in membranous nephropathy were observed. Increase in heparanase activity without changes in superoxide dismutase activity in the granulocytes from patients with lupus nephritis and membranous nephropathy was observed. It may be used as one of the markers of these disease activities.

  10. Tumour-derived GM-CSF promotes granulocyte immunosuppression in mesothelioma patients.

    Science.gov (United States)

    Khanna, Swati; Graef, Suzanne; Mussai, Francis; Thomas, Anish; Wali, Neha; Yenidunya, Bahar Guliz; Yuan, Constance M; Morrow, Betsy; Zhang, Jingli; Korangy, Firouzeh; Greten, Tim F; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Middleton, Gary; De Santo, Carmela; Hassan, Raffit

    2018-03-30

    The cross talk between tumour cells, myeloid cells, and T cells play a critical role in tumour pathogenesis and response to immunotherapies. Although the aetiology of mesothelioma is well understood the impact of mesothelioma on the surrounding immune microenvironment is less well studied. In this study the effect of the mesothelioma microenvironment on circulating and infiltrating granulocytes and T cells is investigated. Tumour and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T cell suppression. Co-cultures of granulocytes, mesothelioma cells, T cells were used to identify the mechanism of T cell inhibition. Analysis of tumours showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T cell proliferation and activation. Characterisation of the blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared to healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of Reactive Oxygen Species (ROS) from granulocytes, resulting in T cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumours express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralising antibody, or ROS inhibition, restored T cell proliferation suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients. Our study presents the mechanism behind the cross-talk between mesothelioma and the immune micro-environment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy. Copyright ©2018, American Association for Cancer Research.

  11. Seasonality in molecular and cytometric diversity of marine bacterioplankton: the reshuffling of bacterial taxa by vertical mixing

    KAUST Repository

    García, Francisca C.

    2015-07-17

    The ’cytometric diversity’ of phytoplankton communities has been studied based on single-cell properties, but the applicability of this method to characterize bacterioplankton has been unexplored. Here, we analysed seasonal changes in cytometric diversity of marine bacterioplankton along a decadal time-series at three coastal stations in the Southern Bay of Biscay. Shannon-Weaver diversity estimates and Bray-Curtis similarities obtained by cytometric and molecular (16S rRNA tag sequencing) methods were significantly correlated in samples from a 3.5-year monthly time-series. Both methods showed a consistent cyclical pattern in the diversity of surface bacterial communities with maximal values in winter. The analysis of the highly resolved flow cytometry time-series across the vertical profile showed that water column mixing was a key factor explaining the seasonal changes in bacterial composition and the winter increase in bacterial diversity in coastal surface waters. Due to its low cost and short processing time as compared to genetic methods, the cytometric diversity approach represents a useful complementary tool in the macroecology of aquatic microbes.

  12. Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis

    DEFF Research Database (Denmark)

    Vindeløv, L L; Hansen, H H; Christensen, I J

    1980-01-01

    Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination and a ...

  13. Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods

    NARCIS (Netherlands)

    M.O. de Jong (Marg); H. Rozemuller (Henk); J.G.J. Bauman (J. G J); J.W.M. Visser (Jan)

    1995-01-01

    textabstractThe main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that

  14. Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2

    Directory of Open Access Journals (Sweden)

    L Postiglione

    2009-06-01

    Full Text Available We investigated the effects of human granulocyte macrophage- colony stimulating factor (GM-CSF on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II, and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by “laddering analysis”, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

  15. Observations on transition of polycythaemia vera into acute or chronic granulocytic leukaemia during treatment with radioactive phosphorus 32P

    International Nuclear Information System (INIS)

    Krasnik, W.

    1975-01-01

    In a group of 172 cases of polycythaemia vera treated with radioactive phosphorus 32 P acute granulocytic leukaemia developed in 3 cases and chronic granulocytic leukaemia in 6 cases. Development of acute granulocytic leukaemia during treatment with radioactive phosphorus for polycythaemia vera may be considered with some probability as a result of leukaemia-inducing action of ionizing radiation. Transition of polycythaemia vera into chronic granulocytic leukaemia seems to a natural outcome of this complex myeloproliferative syndrome in patients with survival prolonged by treatment with 32 P. (author)

  16. Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells

    International Nuclear Information System (INIS)

    Lampariello, F.; Mauro, F.; Uccelli, R.; Spano, M.

    1989-01-01

    An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.18 references

  17. Evaluation of Prognostic Factors Following Flow-Cytometric DNA Analysis after Cytokeratin Labelling: I. Breast Cancer

    Directory of Open Access Journals (Sweden)

    Pauline Wimberger

    2002-01-01

    Full Text Available In gynecologic oncology valid prognostic factors are necessary to estimate the course of disease and to define biologically similar subgroups for analysis of therapeutic efficacy. The presented study is a prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in breast cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17 prior to flow cytometric cell cycle analysis in 327 fresh specimens of primary breast cancer. Univariate analysis in breast cancer detected the prognostic significance of DNA‐ploidy, S‐phase fraction and CV (coefficient of variation of G0G1‐peak of tumor cells for clinical outcome, especially for nodal‐negative patients. Multivariate analysis could not confirm prognostic evidence of DNA‐ploidy and S‐phase fraction. In conclusion, in breast cancer no clinical significance for determination of DNA‐parameters was found.

  18. Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival

    International Nuclear Information System (INIS)

    Welleweerd, J.; Wilder, M.E.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Chinese hamster M3-1 cells were irradiated with several doses of x rays or α particles from 238 Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and α particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods

  19. Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir

    International Nuclear Information System (INIS)

    George, L.S.; Dallas, C.E.; Brisbin, I.L. Jr.; Evans, D.L.

    1991-01-01

    The objective of this study was to detect red blood cell (rbc) DNA abnormalities in male, game-farm mallard ducks as they ranged freely and accumulated 137Cs (radiocesium) from an abandoned nuclear reactor cooling reservoir. Prior to release, the ducks were tamed to enable recapture at will. Flow cytometric measurements conducted at intervals during the first year of exposure yielded cell cycle percentages of DNA (G0/G1, S, G2 + M phases) of rbc, as well as coefficients of variation (CV) in the G0/G1 phase. DNA histograms of exposed ducks were compared with two sets of controls which were maintained 30 and 150 miles from the study site. 137Cs live wholebody burdens were also measured in these animals in a parallel kinetics study, and an approximate steady-state equilibrium was attained after about 8 months. DNA histograms from 2 of the 14 contaminated ducks revealed DNA aneuploid-like patterns after 9 months exposure. These two ducks were removed from the experiment at this time, and when sampled again 1 month later, one continued to exhibit DNA aneuploidy. None of the control DNA histograms demonstrated DNA aneuploid-like patterns. There were no significant differences in cell cycle percentages at any time point between control and exposed animals. A significant increase in CV was observed at 9 months exposure, but after removal of the two ducks with DNA aneuploidy, no significant difference was detected in the group monitored after 12 months exposure. An increased variation in the DNA and DNA aneuploidy could, therefore, be detected in duck rbc using flow cytometric analysis, with the onset of these effects being related to the attainment of maximal levels of 137Cs body burdens in the exposed animals

  20. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Directory of Open Access Journals (Sweden)

    Serkan Yazıcı

    2015-01-01

    Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

  1. Multivariate analysis of flow cytometric data using decision trees

    Directory of Open Access Journals (Sweden)

    Svenja eSimon

    2012-04-01

    Full Text Available Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called 'induction of decision trees' in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees's quality, we used stratified 5-fold cross-validation and chose the 'best' tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.

  2. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

    Science.gov (United States)

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

    2015-03-15

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.

  3. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  4. Immunofluorescence in multiple tissues utilizing serum from a patient affected by systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Piotr Brzezinski

    2012-01-01

    Full Text Available Introduction: Lupus erythematosus is a chronic, inflammatory autoimmune disease that can affect multiple organs. Lupus can affect many parts of the body, especially in systemic lupus erythematosus (SLE; affected tissues may include the joints, skin, kidneys, heart, lungs, blood vessels, and brain. Case report: A 46-year-old female presented with pruritus, photosensitivity and edema of the cheeks of about 2 years duration, and was evaluated by a dermatologist. On examination, multiple telangiectasias were present on the cheeks, with erythema, edema and a malar rash observed. A review of systems documented breathing difficulty and pleuitic pain, joint pain and joint edema, photosensitivity, cardiac dysrhythmia, and periodic pain in the back close to the kidneys. Methods: Skin biopsies for hematoxylin and eosin testing, as well for direct and indirect immunofluorescence were performed, in addition to multiple diagnostic blood tests, chest radiography and directed immunologic testing. Results: The blood testing showed elevated C-reactive protein. Direct and indirect immunofluorescence testing utilizing monkey esophagus, mouse and pig heart and kidney, normal human eyelid skin and veal brain demonstrated strong reactivity to several components of smooth muscle, nerves, blood vessels, skin basement membrane zone and sweat gland ducts and skin meibomian glands. Anti-endomysium antibodies were detected as well as others, especially using FITC conjugated Complement/C1q, FITC conjugated anti-human immunoglobulin IgG and FITC conjugated anti-human fibrinogen. Conclusions: We conclude that both direct and indirect immunofluorescence using several substrates can unveil previously undocumented autoantibodies in multiple organs in lupus erythematosus, and that these findings could be utilized to complement existing diagnostic testing for this disorder.

  5. Lung transit of /sup 111/Indium-labelled granulocytes. Relationship to labelling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Saverymuttu, S.H.; Peters, A.M.; Danpure, H.J.; Reavy, H.J.; Osman, S.; Lavender, J.P. (Hammersmith Hospital, London, England)

    1983-01-01

    The early in vivo distribution of /sup 111/Indium-labelled granulocytes, recorded by dynamic imaging using a gamma camera and computer, varied according to the separation and labelling technique. Following i.v. bolus injection, 4 kinetic patterns could be identified: (A) rapid transit through the pulmonary vasculature, (B) delayed transit through the lung with clearance by about 30 min, (C) complete retention by the lung, for up to 10 min, followed by slow release over a period of 1 to 2 h, (D) delayed transit through the lung with a similar time course to (B) but with subsequent heavy liver uptake. Granulocytes labelled with /sup 111/In-tropolonate and maintained in plasma throughout the labelling procedure, whether injected as a 'pure' (separated by plasma-enriched density gradient centrifugation) or 'crude' (seprated by differential centrifugation) preparation, displayed type A kinetics, thought to most closely represent the normal behaviour of granulocytes. 'Crude' cells labelled in saline with /sup 111/In-acetylacetonate displayed type B kinetics. 'Pure' cells isolated on Percoll-saline and labelled in saline with /sup 111/In-acetylacetonate displayed type C kinetics, thought to represent granulocyte 'stimulation' and/or damage, or type D kientics, thought to represent severe damage. The importance is stressed of labelling granulocytes for kinetic studies with a technique that results in minimal alteration of cell behaviour.

  6. Radiation Effects on Granulocyte Formation and Maturation in Various Species and at Different Levels of Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Fliedner, T. M. [Forschungsgruppe Freiburg, Institut fuer Haematologie der Gesellschaft fuer Strahlenforschung Assoziation mit EURATOM, Freiburg/Breisgau, Federal Republic of Germany (Germany)

    1967-07-15

    Granulocytopenia is one of the well-known consequences of radiation exposure of all or part of the body. It is of concern to the clinician who has to deal with the possible manifestation of bacterial infection that is associated with its development. For the investigator, the time course and pattern of granulocyte changes in the peripheral blood and of their precursors in the bone marrow after radiation may serve to indicate the response of a cell renewal system in general, since its internal structure with a stem-cell pool, a proliferating pool,, a maturation pool and a functioned pool appears to be the same in many other cell renewal systems. Since several of the time parameters of the granulocytic cell renewal system are known as well as the consequences of whole-body irradiation on this system for several species, it may be of interest to this Panel to analyse the radiation effects on granulocytopoiesis. This problem has been the concern of several previous reviews. It is the purpose of this paper to study the following aspects: (a) pattern of development of granulocytopenia as a function of exposure level and of species, (b) comparison of granulocyte maturation in different species as a basis for the analysis of granulocyte depression, and (c) appearance and disappearance of granulocytes with mitotically connected abnormalities as a possible indicator of radiation effects on the proliferative pool.

  7. Effects of irradiation and storage on granulocytes harvested by continuous-flow centrifugation

    International Nuclear Information System (INIS)

    Patrone, F.; Dallegri, F.; Brema, F.; Sacchetti, C.

    1979-01-01

    Five normal subjects were subjected to leukapheresis by continuous-flow-centrifugation (CFC) in the Aminco Celltrifuge. Granulocyte functional capacities were evaluated on the venous blood samples drawn before apheresis and on the cellrich plasma collected by CFC, immediately after collection and after short-term storage at 4degC with or without previous irradiation (1500 rad, 50 rad/min). The CFC technique has been shown to provide cells without functional damage. Irradiation did not appear to influence granulocyte function, as evaluated by in vitro studies. The data demonstrate that granulocytes maintain, even after irradiation, functional activities similar to those found immediately after collection for up to 24 hours of storage at 4degC and exhibit only a moderate loss of function after 48 h. Chemotaxis appears to be the most sensitive detector of cellular damage of stored granulocytes, either irradiated or non-irradiated; this technique may be the most useful for assessment of granulocyte function before transfusion. (author)

  8. Aliphatic alcohol contaminants of illegally produced spirits inhibit phagocytosis by human granulocytes.

    Science.gov (United States)

    Pál, László; Árnyas, Ervin M; Tóth, Béla; Ádám, Balázs; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2013-04-01

    Unregulated production of spirits in many countries leads to products containing appreciable levels of aliphatic alcohols (AAs) and is the main source of human exposure to these substances worldwide. Previous studies have confirmed that alcohol abuse can lead to ethanol-induced immunosuppression and thereby increased susceptibility to infectious diseases. Granulocytes, as professional phagocytic cells, play a crucial role in engulfment and killing of pathogenic microorganisms. Thus, a decrease in their phagocytic activity has been invoked as a factor in the impaired antimicrobial defense observed in alcoholics. However, AAs consumed as contaminants of illicit spirits may also influence phagocytosis, thereby contributing to a further decrease in microbicidal activity but, so far, this has not been studied. Therefore, the aim of this study was to measure granulocyte phagocytosis following treatment of granulocytes with those higher alcohols found in illegal spirits. Granulocytes were isolated from human peripheral blood. Then phagocytosis of opsonized zymosan particles by granulocytes treated with AAs individually and in combination was determined. These alcohols inhibited phagocytosis in a concentration-dependent manner and at lower concentrations when combined than when tested individually. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit phagocytosis in a clinically meaningful way in episodic heavy drinkers.

  9. Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

    Science.gov (United States)

    Bakherad, Hamid; Gargari, Seyed Latif Mousavi; Sepehrizadeh, Zargham; Aghamollaei, Hossein; Taheri, Ramezan Ali; Torshabi, Maryam; Yazdi, Mojtaba Tabatabaei; Ebrahimizadeh, Walead; Setayesh, Neda

    2017-09-01

    It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models. Copyright © 2017. Published by Elsevier Masson SAS.

  10. Development of immunofluorescence colony staining (IFC) for detection of Xanthomonas campestris pv. vesicatoria and Clavibacter michiganensis subsp michiganensis in tomato seeds

    NARCIS (Netherlands)

    Nemeth, J.; Vuurde, van J.W.L.

    2006-01-01

    Immunofluorescence colony-staining (IFC) is based on sample pour plating in combination with immunofluorescence staining for recognition of the target colony. IFC was optimised for detecting Xanthomonas campestris pv. vesicatoria (Xcv) and Clavibacter michiganensis subsp. michiganensis (Cmm) in

  11. Diagnostic value of (111)In-granulocyte scintigraphy in patients with fever of unknown origin

    DEFF Research Database (Denmark)

    Kjaer, Andreas; Lebech, Anne-Mette

    2002-01-01

    111In-granulocyte scintigraphy is often used as a diagnostic tool in patients with fever of unknown origin (FUO). However, its diagnostic performance has been studied in only a limited number of investigations, with most having been published more than 10 y ago; in addition, a broad range...... of sensitivities and specificities has been reported. Therefore, the aim of this study was to investigate the diagnostic value of granulocyte scintigraphy in patients fulfilling the criteria of FUO. Also studied was whether increased peripheral leukocyte count or C-reactive protein (CRP) level could be used...... to select patients for scintigraphy to raise the diagnostic value. METHODS: For 31 patients with true FUO who underwent granulocyte scintigraphy at a third-line referral hospital between 1995 and 2000, the files and scintigraphy findings were reviewed retrospectively to test the ability of scintigraphy...

  12. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    International Nuclear Information System (INIS)

    Renò, Filippo; Rizzi, Manuela; Pittarella, Pamela; Carniato, Fabio; Olivero, Francesco; Marchese, Leonardo

    2013-01-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine. (paper)

  13. Autologous /sup 111/In-oxine-labeled granulocytes in Yersinia infections

    Energy Technology Data Exchange (ETDEWEB)

    Becker, W.; Boerner, W.; Fischbach, W.

    1985-04-01

    Autologous /sup 111/In-oxine-labeled granulocytes have proved to be valuable for the localization of inflammatory bowel diseases, especially Crohn's disease and ulcerative colitis. Other rare inflammatory bowel diseases also yield positive /sup 111/In scans. One case of Yersinia infection of the terminal ileum (Yersinia enterocolitica) showing an accumulation of /sup 111/In-oxine-labeled granulocytes 0.5, 4, and 24 h after the reinjection of the labeled cells is described. The 4-day fecal excretion of /sup 111/In-oxine granulocytes showed a slight inflammatory activity of the terminal ileum. One negative scan is reported in a cotrimoxazole-treated patient with Yersinia infection.

  14. Nail unit in collagen vascular diseases: A clinical, histopathological and direct immunofluorescence study

    Directory of Open Access Journals (Sweden)

    Nabil P

    2006-01-01

    Full Text Available Background: Abnormalities of the nail unit are common in patients with connective tissue diseases. Clinical examination of the nail unit, coupled with biopsy of proximal nail fold offers an additional advantage in the diagnosis. Purpose: Our aim was to record clinical changes of the nail unit in connective tissue diseases and to study the histopathological (both H and E and periodic acid Schiff and direct immunofluorescence (DIF findings of nail-fold biopsy. Materials and Methods: Thirty-eight confirmed cases connective tissue diseases attending skin OPD were enrolled in the study. After detailed clinical examination of the nail unit, a crescentric biopsy was taken from the proximal nail fold (PNF. Histopathological and DIF studies were was carried out. Findings: Nail changes could be demonstrated in 65% connective tissue diseases. Specific histopathological (H and E and immunofluorescence findings were also encountered in many patients. Conclusion: Clinical examination of the nail unit offers additional clue in the diagnosis of connective tissue diseases. Though DIF of PNF biopsy is useful in the diagnosis, it is not an ideal site for H and E study, as the yield is very low. Limitations: Lack of adequate comparison group and non-utilization of capillary microscopy for the detection of nail fold capillary abnormalities.

  15. Kidney lesions in Rocky Mountain spotted fever: a light-, immunofluorescence-, and electron-microscopic study.

    Science.gov (United States)

    Bradford, W. D.; Croker, B. P.; Tisher, C. C.

    1979-01-01

    The essential pathologic lesion in Rocky Mountain spotted fever (RMSF) is a vasculitis that may involve the kidneys as well as the heart, brain, skin, and subcutaneous tissues. Histopathologic information concerning the response of the kidneys in RMSF is rather limited, however. In this study renal tissue from 17 children who died of RMSF was examined by light, electron, and immunofluorescence microscopy. A lymphocytic or mixed inflammation, or both, involving vessels and interstitium of the kidney was found in all patients. In addition, 10 patients had histologic evidence of acute tubular necrosis, and another 3 had glomerular lesions consisting of focal segmental tuft necrosis or increased cellularity secondary to neutophilic infiltration, or both. Immunofluorescence- and electron-microscopic studies failed to demonstrate immune-complex deposition within glomeruli, a finding that suggests that immunoglobulin and classic immune complexes were not involved in the pathogenesis of the renal lesions at the time of death. These findings suggest the possibility that the pathogenesis of the renal lesion in RMSF may be due to a direct action of the organism (Rickettsia rickettsii) on the vessel wall. Images Figure 2 Figure 1 PMID:525676

  16. EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

    Science.gov (United States)

    Krause, C; Ens, K; Fechner, K; Voigt, J; Fraune, J; Rohwäder, E; Hahn, M; Danckwardt, M; Feirer, C; Barth, E; Martinetz, T; Stöcker, W

    2015-04-01

    Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  17. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

    Directory of Open Access Journals (Sweden)

    Sandrine Prost

    Full Text Available The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family. Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705, Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.

  18. Original Approach for Automated Quantification of Antinuclear Autoantibodies by Indirect Immunofluorescence

    Directory of Open Access Journals (Sweden)

    Daniel Bertin

    2013-01-01

    Full Text Available Introduction. Indirect immunofluorescence (IIF is the gold standard method for the detection of antinuclear antibodies (ANA which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE based on the calculation of a fluorescence index (FI. Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92, even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ=0.80, P<0.0001. Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. Conclusion. Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization.

  19. CD18 expression in granulocytes infiltrating the vitreous fluid in patients with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Qi; Zhu; Hu-Ping; Song

    2015-01-01

    AIM: To assess the levels of CD18 on the surface of granulocytes infiltrating the vitreous fluid in patients with diabetic retinopathy(DR).METHODS: Vitreous samples from twelve patients with non-proliferative DR with significant macula edema(group A), 33 patients with proliferative DR(grade 3 as group B, n =14, and, grade 4 as group C, n =19) were obtained during pars plana vitrectomy. Vitreous samples from 12 patients with macular hole as controls(group D)were analyzed together. The infiltrating of granulocytes and its surface level of CD18 were measured by flow cytometry. The level of CD18 was presented as the mean channel fluorescence(MCF) on a logarithmic scale. RESULTS: Granulocytes were detected in 6 of 12 vitreous samples from group A, 9 of 14 from group B, 15 of 19 from group C, and none of 12 from group D. MCF of CD18 on granulocytes from groups A, B, and C were2.978 ±1.446, 3.201 ±0.692, and 4.072 ±0.837, respectively.The difference was significant(F =4.354, P =0.021).Subjects with more severe DR were more likely to have a higher level of CD18 MCF(trend test, 掊2=7.351, P =0.007).CD18 MCF was significantly associated with the development of DR(r =0.46, P =0.005 and β =0.147, P =0.035).CONCLUSION: Our results confirm the presence of granulocytes and the elevated levels of CD18 on the surface of them in the vitreous fluid from DR patients.These results may provide indirect evidence shown that granulocytes activation also has occurred in the retinal local compared to non-DR control.

  20. Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol.

    Science.gov (United States)

    Vian, Alex M; Higgins, Adam Z

    2014-02-01

    Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van't Hoff model. This yielded an isotonic cell volume of 378 μm(3) and an osmotically inactive volume of 165 μm(3). To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37°C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21°C of 0.18 μmatm(-1)min(-1). The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21°C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. /sup 99/Tcsup(m)-HMPAO labelled leucocytes: comparison with /sup 111/In-tropolonate labelled granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M.; Roddie, M.E.; Zacharopoulos, G.P.; George, P.; Stuttle, A.W.J.; Lavender, J.P.; Danpure, H.J.; Osman, S.

    1988-06-01

    The lipophilic complex, /sup 99/Tcsup(m)-hexamethylpropyleneamine oxime (HMPAO) is an efficient leucocyte label, and labels granulocytes with more stability than mononuclear leucocytes. The recovery of /sup 99/Tcsup(m)-HMPAO granulocytes was similar to /sup 111/In-labelled granulocytes isolated and labelled in plasma using tropolone. The Tsub(1/2) of /sup 99/Tcsup(m)-HMPAO labelled granulocytes in blood was less than that of /sup 111/In-labelled granulocytes. The initial biodistribution of /sup 99/Tcsup(m)-labelled leucocytes was similar to /sup 111/In-labelled granulocytes, with a rapid initial lung transit, prominent splenic activity, bone marrow activity and minimal hepatic activity, although, unlike /sup 111/In, /sup 99/Tcsup(m) activity was also seen in urine, occasionally in the gallbladder, and, from about 4 h, consistently in the colon. Bone marrow activity was particularly prominent with /sup 99/Tcsup(m). About 6% of /sup 99/Tcsup(m) was excreted in the faeces up to 48 h after injection, and about 17% in urine up to 24 h. The time-activity curves of reticuloendothelial activity up to 24 h were broadly similar for the two labelled cell preparations. Clinical information given by the two agents was similar in 27 of 30 patients who received both. We conclude that with respect to granulocyte kinetics and clinical data, /sup 99/Tcsup(m)-HMPAO labelled leucocytes are comparable with /sup 111/In-tropolonate labelled granulocytes.

  2. Granulocytic sarcoma presenting with necrotic cervical lymph nodes as an initial manifestation of childhood leukaemia: imaging features

    Energy Technology Data Exchange (ETDEWEB)

    An, Sang Bu; Cheon, Jung-Eun; Kim, In-One; Kim, Woo Sun [Seoul National University College of Medicine, Department of Radiology, Seoul (Korea); Seoul National University Medical Research Center, Institute of Radiation Medicine, Seoul (Korea); Ahn, Hyo Seop; Shin, Hee Young; Kang, Hyoung Jin; Yeon, Kyung Mo [Seoul National University College of Medicine, Department of Pediatrics, Cancer Research Institute, Seoul (Korea)

    2008-06-15

    We present two cases of granulocytic sarcoma of the cervical lymph nodes with central necrosis as an initial manifestation of childhood leukaemia, focusing on the imaging features. Recognition of the CT and MR imaging findings of granulocytic sarcoma involving the cervical lymph nodes assists the differential diagnosis of noninfective lymphadenopathy in children. (orig.)

  3. Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

    Science.gov (United States)

    Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

    2003-03-14

    Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

  4. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  5. Diagnostic value of (111)In-granulocyte scintigraphy in patients with fever of unknown origin

    DEFF Research Database (Denmark)

    Kjaer, Andreas; Lebech, Anne-Mette

    2002-01-01

    111In-granulocyte scintigraphy is often used as a diagnostic tool in patients with fever of unknown origin (FUO). However, its diagnostic performance has been studied in only a limited number of investigations, with most having been published more than 10 y ago; in addition, a broad range...... and specificity in cases of FUO, when one takes into account that (111)In-granulocyte scintigraphy is not a first-line test. The high predictive value of a scintigram showing negative findings may be especially valuable for ruling out an infectious cause of FUO. Neither peripheral leukocyte count nor CRP levels...

  6. The Granulocyte-colony stimulating factor has a dual role in neuronal and vascular plasticity

    Directory of Open Access Journals (Sweden)

    Stephanie eWallner

    2015-08-01

    Full Text Available Granulocyte-colony stimulating factor (G-CSF is a growth factor that has originally been identified several decades ago as a hematopoietic factor required mainly for the generation of neutrophilic granulocytes, and is in clinical use for that. More recently, it has been discovered that G-CSF also plays a role in the brain as a growth factor for neurons and neural stem cells, and as a factor involved in the plasticity of the vasculature. We review and discuss these dual properties in view of the neuroregenerative potential of this growth factor.

  7. [Reversibility of the leukocyte activation state studied in a model of endogenous pyrogen formation by granulocytes].

    Science.gov (United States)

    Rybakina, E G; Sorokin, A V

    1980-08-01

    The pyrogen-releasing capacity of rabbit exudate granulocytes can be temporarily suppressed during incubation in the whole plasma and then recovered during cell transfer into 0.15 M NaCl or stimulation with the bacterial lipopolysaccharide, pyrogenal. The inhibitors of protein synthesis added to the granulocytes when they are being transferred from plasma to 0.15 M NaCl do not suppress the pyrogen release. The inhibitory action of the whole plasma on the pyrogen release is due to the presence in it of potassium and calcium ions. The inhibitory factors of plasma reversibly suppress the pyrogen release but do not eliminate the leukocyte activation.

  8. Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

    Science.gov (United States)

    Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

    2008-09-15

    Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

  9. THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

    NARCIS (Netherlands)

    VRIELING, EG; DRAAIJER, A; VANZEIJL, WJM; PEPERZAK, L; GIESKES, WWC; VEENHUIS, M; Zeijl, Wilhelmus J.M. van

    Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by

  10. Comparative determination of the rheumatic factor by means of agglutination, immunofluorescence and radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Jaeger, L.; Storz, H.; Hein, G.; Schlenvoigt, G. (Friedrich-Schiller-Universitaet, Jena (German Democratic Republic). Bereich Medizin)

    1982-01-01

    The rheumatic factor (RF) was determined by means of agglutination, immunofluorescence (IF) test and radioimmunoassay (RIPEGA) in random groups of 56 patients with rheumatoid arthritis (RA), 13 patients with seronegative RA and 39 patients with psoriasis arthropathica. All three methods are of equal value with regard to the number of positive results. Further classification of seronegative patients, i.e. patients with a negative agglutination reaction and the clinical symptoms of RA is possible with the IF method and, above all, by means of RIPEGA. But because of the comprehensive test devices the two methods are only an alternative. Titer differences are attributed to the different indication principles and the immunological heterogeneity of RF. An improvement of the diagnosis of activity was not possible.

  11. Comparative determination of the rheumatic factor by means of agglutination, immunofluorescence and radioimmunoassay

    International Nuclear Information System (INIS)

    Jaeger, L.; Storz, H.; Hein, G.; Schlenvoigt, G.

    1982-01-01

    The rheumatic factor (RF) was determined by means of agglutination, immunofluorescence (IF) test and radioimmunoassay (RIPEGA) in random groups of 56 patients with rheumatoid arthritis (RA), 13 patients with seronegative RA and 39 patients with psoriasis arthropathica. All three methods are of equal value with regard to the number of positive results. Further classification of seronegative patients, i.e. patients with a negative agglutination reaction and the clinical symptoms of RA is possible with the IF method and, above all, by means of RIPEGA. But because of the comprehensive test devices the two methods are only an alternative. Titer differences are attributed to the different indication principles and the immunological heterogeneity of RF. An improvement of the diagnosis of activity was not possible. (author)

  12. Staining pattern classification of antinuclear autoantibodies based on block segmentation in indirect immunofluorescence images.

    Directory of Open Access Journals (Sweden)

    Jiaqian Li

    Full Text Available Indirect immunofluorescence based on HEp-2 cell substrate is the most commonly used staining method for antinuclear autoantibodies associated with different types of autoimmune pathologies. The aim of this paper is to design an automatic system to identify the staining patterns based on block segmentation compared to the cell segmentation most used in previous research. Various feature descriptors and classifiers are tested and compared in the classification of the staining pattern of blocks and it is found that the technique of the combination of the local binary pattern and the k-nearest neighbor algorithm achieve the best performance. Relying on the results of block pattern classification, experiments on the whole images show that classifier fusion rules are able to identify the staining patterns of the whole well (specimen image with a total accuracy of about 94.62%.

  13. Multiplexed Immunofluorescence Reveals Potential PD-1/PD-L1 Pathway Vulnerabilities in Craniopharyngioma.

    Science.gov (United States)

    Coy, Shannon; Rashid, Rumana; Lin, Jia-Ren; Du, Ziming; Donson, Andrew M; Hankinson, Todd C; Foreman, Nicholas K; Manley, Peter E; Kieran, Mark W; Reardon, David A; Sorger, Peter K; Santagata, Sandro

    2018-03-02

    Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous (ACP) and papillary (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the PD-1/PD-L1 immune checkpoint pathway in ACP and PCP. We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence (t-CyCIF) to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1. All ACP (15±14% of cells, n=23, average±S.D.) and PCP (35±22% of cells, n=18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst-lining. In PCP, PD-L1 was highly-expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mTOR and MAPK signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T-cells. ACP exhibit PD-L1 expression in the tumor cyst-lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.

  14. An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

    Science.gov (United States)

    Koch, Peter; Herzig, Stefan; Matthes, Jan

    Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Ca v 1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Ca v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca v 1.2 protein and a correct Ca v 1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides

    Directory of Open Access Journals (Sweden)

    Alessandra Dellavance

    2013-06-01

    Full Text Available INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP, centromeric protein F (CENP-F, proliferating cell nuclear antigen (PCNA, cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase, nuclear mitotic apparatus protein 1 (NuMA-1 and nuclear mitotic apparatus protein 2 (NuMA-2. CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

  16. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

    Science.gov (United States)

    Knapp, W; Strobl, H; Majdic, O

    1994-12-15

    New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

  17. Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans

    Directory of Open Access Journals (Sweden)

    Jesús M. Egido

    1997-12-01

    Full Text Available We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripherial blood (HCPB, using commercially available phycoerythrin-conjugated latex particles of 1µm diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984 standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripherial blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB.Se da cuenta de un método rápido para la cuantización del flujo citométrico de la fagocitosis en sangre periférica completamente heparinizada (HCPB, mediante la utilización de partículas de látex phycoerythrin-conjugadas de 1µm de diámetro disponibles comercialmente. El método es más rápido y presenta mayor reproducibilidad que la técnica estandar de Bjerknes' (1984 utilizando propidium iodide-teñida Candida albicans, aplicada convencionalmente a la capa leucocitica de sangre periférica pero modificada por HCPB. Tambien damos cuenta de una modificación de Bjerknes' Intracellular Killing Test para permitir su aplicación a HCPB.

  18. Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice

    International Nuclear Information System (INIS)

    Yishai, Yitzhak; Fixler, Dror; Cohen-Kashi, Meir; Zurgil, Naomi; Deutsch, Mordechai

    2003-01-01

    The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP

  19. A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

    Directory of Open Access Journals (Sweden)

    Gentry-Nielsen Martha J

    2006-07-01

    Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

  20. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Coignoul Freddy

    2007-09-01

    Full Text Available Abstract Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

  1. Determination of chitin content in fungal cell wall: an alternative flow cytometric method.

    Science.gov (United States)

    Costa-de-Oliveira, Sofia; Silva, Ana P; Miranda, Isabel M; Salvador, Alexandre; Azevedo, Maria M; Munro, Carol A; Rodrigues, Acácio G; Pina-Vaz, Cidália

    2013-03-01

    The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. Copyright © 2013 International Society for Advancement of Cytometry.

  2. Ovarian granulocytic sarcoma: a case report and magnetic resonance imaging findings

    International Nuclear Information System (INIS)

    Pereira, Licia Pacheco; Monte, Hipolito

    2008-01-01

    Granulocytic sarcoma (chloroma) is a tumor consisting of myeloid precursors in an extramedullary site. It is complication of both acute and chronic myelogenous leukemias. Although the lesion can occur at any site, ovarian involvement is rare. We report a case of ovary tumor associated with acute myeloid leukaemia and its imaging appearance on magnetic resonance. (author)

  3. Type 3 Deiodinase Is Highly Expressed in Infiltrating Neutrophilic Granulocytes in Response to Acute Bacterial Infection

    NARCIS (Netherlands)

    Boelen, Anita; Boorsma, Jeffrey; Kwakkel, Joan; Wieland, Catharina W.; Renckens, Rosemarijn; Visser, Theo J.; Fliers, Eric; Wiersinga, Wilmar M.

    2008-01-01

    Background: Macrophages and polymorphonuclear cells (PMNs) play an important role in the first line of defense against bacteria by infiltrating the infected organ in order to clear the harmful pathogen. Our earlier studies showed that granulocytes express type 3 deiodinase (D3) when activated during

  4. Granulocyte Colony-Stimulating Factor in the Treatment of Acute Radiation Syndrome: A Concise Review

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Komůrková, Denisa; Hoferová, Zuzana

    2014-01-01

    Roč. 19, č. 4 (2014), s. 4770-4778 ISSN 1420-3049 R&D Projects: GA ČR(CZ) GAP303/11/0128 Institutional support: RVO:68081707 Keywords : granulocyte colony-stimulating factor * radiation accident s * acute radiation syndrome Subject RIV: BO - Biophysics Impact factor: 2.416, year: 2014

  5. Mitochondrial Fragmentation in Aspergillus fumigatus as Early Marker of Granulocyte Killing Activity

    Science.gov (United States)

    Ruf, Dominik; Brantl, Victor; Wagener, Johannes

    2018-01-01

    The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. PMID:29868488

  6. Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

    NARCIS (Netherlands)

    Verburg-van Kemenade, B.M.L.; Weyts, F.A.A.; Debets, R.; Flik, G.

    1995-01-01

    Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig− lymphocytes as well as of the

  7. Effect of inflammatory serum of 14C-glucosamine incorporation into bone marrow granulocytes in vitro

    International Nuclear Information System (INIS)

    Evans, W.H.; Wilson, S.M.; Torres, A.R.; Peterson, E.A.; Mage, M.G.

    1979-01-01

    As a preliminary approach to developing a biochemical assay for detecting humoral regulators of granulocyte maturation in the normal and inglammatory states, studies were carried out on the effects of normal inflammatory sera on the incorporation of 14 C-glucosamine into the glycoproteins of bone marrow granulocytes in vitro. We observed that, relative to normal serum, inflammatory serum had a marked stimulatory effect on 14 C-glucosamine incorporation into these glycoproteins. This property of inflammatory serum reached a maximum at about 8 h after the initiation of inflammation in vivo and preceded the maximum increase in the mitotic activity of granulocyte precursors in the marrow by 18 h. It was also found that normal serum contains both dialyzable and heat-sensitive nondialyzable factors that inhibit 14 C-glucosamine incorporation into bone marrow granulocytes in vitro. Data are presented which indicate that the stimulatory effect of inflammatory serum is most likely due to a nondialyzable factor which is capable of blocking the effect of the inhibitors present in normal serum. (author)

  8. Comparison of new immunofluorescence method for detection of soy protein in meat products with immunohistochemical, histochemical, and ELISA methods

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2014-01-01

    Full Text Available Soy proteins are commonly used in the food industry thanks to their technological properties. However, soy is, along with cow’s milk, eggs, wheat, peanuts, tree nuts, fish, crustaceans, and molluscs, responsible for around 90% of food allergies, and is also one of the foodstuffs that can cause anaphylaxis. The aim of this work was to compare the immunofluorescence method for the detection of soy protein in meat products purchased from the retail market with other microscopic methods (immunohistochemical and histochemical, with the ELISA reference method and with the confirmatory results. Within the research, 127 meat products purchased in the retail network were examined using the immunofluorescence method used for the detection of soy protein. The method was compared to Enzyme-Linked ImmunoSorbent Assay (ELISA, immunohistochemical, and histochemical methods. According to McNemar’s test, non-compliance between the immunofluorescence method and immunohistochemical method was low. In addition, a significant difference between the fluorescence method and ELISA (P P < 0.01 was found. The immunofluorescence method was also compared with confirmatory results. According to McNemar’s test, non-compliance between the immunofluorescence method and confirmatory results was low. The results showed the possibilities of this new method to detect the content of soy protein in meat products.

  9. Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

    Science.gov (United States)

    Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

    2013-01-01

    Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pknowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

  10. Role of opsonins in clinical response to granulocyte transfusion in granulocytopenic patients

    International Nuclear Information System (INIS)

    Keusch, G.T.; Ambinder, E.P.; Kovacs, I.; Goldberg, J.D.; Phillips, D.M.; Holland, J.F.

    1982-01-01

    Fifty febrile severely granulocytopenic patients were given four daily transfusions of 2.2 X 10(10) normal donor granulocytes. Twenty-three responded clinically, although both responders and nonresponders were similar in clinical characteristics at the outset. This study examines the relation between serum opsonic activity before initiation of granulocyte administration and clinical response. Opsonic activity to three test organisms (Escherichia coli 286 and ON 2, and Staphylococcus aureus) and to 15 blood stream isolates from 14 patients was measured as serum-dependent uptake of heat-killed 14 C-labeled bacteria by normal donor leukopheresis granulocytes in an in vitro assay and compared with results obtained with a standard normal serum in each assay. At a concentration of 8 percent serum, all patient groups were equivalent to standard for the three test organisms. When rate-limiting concentrations of serum were employed, opsonic activity remained similar to standard for S. aureus in all patient groups and for the two E. coli strains in responders. In contrast, opsonins for E. coli decreased to 41 to 50 percent of standard in nonresponders. When patients with proved infection were separately analyzed, opsonin activity for E. coli was significantly greater in responders than nonresponders. Eight of 10 patients with 75 percent or greater of standard for opsonic activity against their own blood stream isolates also responded, whereas zero of four with less than 75 percent of standard had a favorable outcome. These results indicate that serum opsonic activity may be a determinant of clinical response to granulocyte transfusion in infected granulocytopenic patients. We conclude that opsonic activity should be assessed in such patients before granulocyte administration and suggest a trial of plasma infusion in opsonin-deficient patients

  11. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

    International Nuclear Information System (INIS)

    Pai, J.K.; Siegel, M.I.; Egan, R.W.; Billah, M.M.

    1988-01-01

    There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration

  12. Tumor-Derived G-CSF Facilitates Neoplastic Growth through a Granulocytic Myeloid-Derived Suppressor Cell-Dependent Mechanism

    Science.gov (United States)

    Waight, Jeremy D.; Hu, Qiang; Miller, Austin; Liu, Song; Abrams, Scott I.

    2011-01-01

    Myeloid-derived suppressor cells (MDSC) are induced under diverse pathologic conditions, including neoplasia, and suppress innate and adaptive immunity. While the mechanisms by which MDSC mediate immunosuppression are well-characterized, details on how they develop remain less understood. This is complicated further by the fact that MDSC comprise multiple myeloid cell types, namely monocytes and granulocytes, reflecting diverse stages of differentiation and the proportion of these subpopulations vary among different neoplastic models. Thus, it is thought that the type and quantities of inflammatory mediators generated during neoplasia dictate the composition of the resultant MDSC response. Although much interest has been devoted to monocytic MDSC biology, a fundamental gap remains in our understanding of the derivation of granulocytic MDSC. In settings of heightened granulocytic MDSC responses, we hypothesized that inappropriate production of G-CSF is a key initiator of granulocytic MDSC accumulation. We observed abundant amounts of G-CSF in vivo, which correlated with robust granulocytic MDSC responses in multiple tumor models. Using G-CSF loss- and gain-of-function approaches, we demonstrated for the first time that: 1) abrogating G-CSF production significantly diminished granulocytic MDSC accumulation and tumor growth; 2) ectopically over-expressing G-CSF in G-CSF-negative tumors significantly augmented granulocytic MDSC accumulation and tumor growth; and 3) treatment of naïve healthy mice with recombinant G-CSF protein elicited granulocytic-like MDSC remarkably similar to those induced under tumor-bearing conditions. Collectively, we demonstrated that tumor-derived G-CSF enhances tumor growth through granulocytic MDSC-dependent mechanisms. These findings provide us with novel insights into MDSC subset development and potentially new biomarkers or targets for cancer therapy. PMID:22110722

  13. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario

    Directory of Open Access Journals (Sweden)

    Sebastian Wendy

    2010-07-01

    Full Text Available Background: Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specific auto antibodies that have so far been associated with specific rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA patterns with the immunoprofile in the Indian population to date. Aims: To understand a definite association between ANA patterns and specific antibodies in the serum in the Indian study population and to document similarities / differences with the West. Settings and Design: This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunofluorescence method and by immunoline methods. Materials and Methods: Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunofluorescence (IIF method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp - 2010 / liver biochip (Monkey (EUROIMMUN AG. The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2 along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. Results: The antinuclear antibody indirect immunofluorescence (ANA - IIF patterns obtained

  14. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

    DEFF Research Database (Denmark)

    Porse, Bo T; Bryder, David; Theilgaard-Mönch, Kim

    2005-01-01

    dissociate the ability of C/EBP alpha to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid...... progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation...

  15. Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.

    Science.gov (United States)

    King, M A; Wu, Q X; Donovan, G R; Baldo, B A

    1998-08-01

    Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

  16. Immunofluorescence pattern of antinuclear antibody and its association with autoantibody profile in systemic lupus erythematosus

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    Sadia Sharmin

    2016-08-01

    Full Text Available Background: Antinuclear antibody (ANA is useful in the diagnosis of systemic lupus erythematosus (SLE. Association of specific autoantibodies with the immunofluorescence pattern of ANA in SLE as noted in Western literature has been taken as reference in all over the world. However, in Bangladesh such research work or data correlating the autoantibodies and their ANA patterns is inadequate. Objective: To identify an association between immunofluorescence patterns of antinuclear antibody on HEp-2 cell and more specific antinuclear reactivities (e.g. anti-dsDNA and anti-extractable nuclear antigen in the serum samples of SLE patients.Methods: Serum samples of 37 SLE patients who were diagnosed by ARA (American Rheumatism Association classification criteria and laboratory tests, attending at lupus clinic of Bangabandhu Sheikh Mujib Medical University (BSMMU during the study period of six months were subjected for ANA testing by Indirect Imrnunofluorescence (IIF on HEp-2 cell, anti-dsDNA by ELISA and anti- extractable nuclear antigen (anti-ENA by Dot Immunoblot. Dot blot strips were tested for anti-Sm, anti-RNP, anti-SSA/Ro, and anti-SSB/La. Results: Out of 37 SLE patients 32 (86.5% cases were ANA positive by IIF on HEp-2 cell. ANA positive sera exhibited three fluorescence patterns such as speckled (43.7%, peripheral (34.3% and homogenous pattern (21.8%. Peripheral pattern (100% was strongly associated with anti-dsDNA (p<0.05 and homogenous pattern (85.7% was also predominantly associated with anti-dsDNA (p<0.05. Speckled pattern (85.6% was significantly associated with anti-ENA (p<0.05. Anti-dsDNA was positive in 75% of SLE cases and majority (45.8% of which showed peripheral pattern whereas anti-ENA was positive in 48.6% cases and majority (70.5% of which showed speckled pattern. The most commonly identified antinuclear autoreactivity was directed towards anti-RNP (22.2% then anti-Sm (16.6%, anti-SSA (16.6% and anti-SSB (11.1 %. Multiple anti

  17. Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

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    O. M. Barth

    1988-06-01

    Full Text Available The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.A presença de antígeno viral em cortes de tecidos humanos fixados em formol e emblocados em parafina foi demonstrada pela digestão com tripsina foi demonstrada pela ingestão com tripsina seguida de imunofluorescência direta ou indireta. Os espécimens podem ser utilizados para diagnoses retrospectivas. A técnica da imunofluorescência deve ser adaptada à infecção viral suspeita segundo diagnosie histopatológica prévia. Os parâmetros para a digestão do tecido pela tripsina, relacionados à concentração, duração de atuação e temperatura, expõem diferentes antígenos virais e devem ser previamente testados para cada sistema a ser estabelecido. Uma digestão mais intensa deve ser aplicada para a detecção do vírus do sarampo em tecido pulmonar do que para adenovírus ou vírus respiratório sincicial no mesmo tecido. Por outro lado, o vírus da febre amarela em tecido de fígado necessita de uma digestão mais fraca.

  18. A flow-cytometric gram-staining technique for milk-associated bacteria.

    Science.gov (United States)

    Holm, Claus; Jespersen, Lene

    2003-05-01

    A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.

  19. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

    2017-10-23

    Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

  20. Genome size variation among and within Camellia species by using flow cytometric analysis.

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    Hui Huang

    Full Text Available BACKGROUND: The genus Camellia, belonging to the family Theaceae, is economically important group in flowering plants. Frequent interspecific hybridization together with polyploidization has made them become taxonomically "difficult taxa". The DNA content is often used to measure genome size variation and has largely advanced our understanding of plant evolution and genome variation. The goals of this study were to investigate patterns of interspecific and intraspecific variation of DNA contents and further explore genome size evolution in a phylogenetic context of the genus. METHODOLOGY/PRINCIPAL FINDINGS: The DNA amount in the genus was determined by using propidium iodide flow cytometry analysis for a total of 139 individual plants representing almost all sections of the two subgenera, Camellia and Thea. An improved WPB buffer was proven to be suitable for the Camellia species, which was able to counteract the negative effects of secondary metabolite and generated high-quality results with low coefficient of variation values (CV <5%. Our results showed trivial effects on different tissues of flowers, leaves and buds as well as cytosolic compounds on the estimation of DNA amount. The DNA content of C. sinensis var. assamica was estimated to be 1C = 3.01 pg by flow cytometric analysis, which is equal to a genome size of about 2940 Mb. CONCLUSION: Intraspecific and interspecific variations were observed in the genus Camellia, and as expected, the latter was larger than the former. Our study suggests a directional trend of increasing genome size in the genus Camellia probably owing to the frequent polyploidization events.

  1. Flow cytometric detection of micronuclei by combined staining of DNA and membranes

    International Nuclear Information System (INIS)

    Wessels, J.M.; Nuesse, M.

    1995-01-01

    A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs

  2. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    Science.gov (United States)

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  3. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Science.gov (United States)

    Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  4. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  5. Flow cytometric chemosensitivity assay using JC‑1, a sensor of mitochondrial transmembrane potential, in acute leukemia.

    Science.gov (United States)

    Yokosuka, Tomoko; Goto, Hiroaki; Fujii, Hisaki; Naruto, Takuya; Takeuchi, Masanobu; Tanoshima, Reo; Kato, Hiromi; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Yokota, Shumpei

    2013-12-01

    The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003). This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

  6. Ligand receptor dynamics at streptavidin-coated particle surfaces: A flow cytometric and spectrofluorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Buranda, T. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Univ. of New Mexico, Albuquerque, NM (United States); Jones, G.M. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States); Nolan, J.P.; Keij, J. [Los Alamos National Labs., NM (United States); Lopez, G.P. [Univ. of New Mexico, Albuquerque, NM (United States); Sklar, L.A. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Los Alamos National Lab., NM (United States)

    1999-04-29

    The authors have studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 {micro}m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. They have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. The authors estimate the site number to be {approx}1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is {approx}(1.3 {+-} 0.3) {times} 10{sup 7} 1{sup {minus}1} S{sup {minus}1}. The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s{sup {minus}1} shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

  7. Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

    Science.gov (United States)

    Dolz, María; O'Connor, José-Enrique; Lequerica, Juan L

    2004-10-01

    The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.

  8. Intestinal intraepithelial lymphocyte cytometric pattern is more accurate than subepithelial deposits of anti-tissue transglutaminase IgA for the diagnosis of celiac disease in lymphocytic enteritis.

    Directory of Open Access Journals (Sweden)

    Fernando Fernández-Bañares

    Full Text Available BACKGROUND & AIMS: An increase in CD3+TCRγδ+ and a decrease in CD3- intraepithelial lymphocytes (IEL is a characteristic flow cytometric pattern of celiac disease (CD with atrophy. The aim was to evaluate the usefulness of both CD IEL cytometric pattern and anti-TG2 IgA subepithelial deposit analysis (CD IF pattern for diagnosing lymphocytic enteritis due to CD. METHODS: Two-hundred and five patients (144 females who underwent duodenal biopsy for clinical suspicion of CD and positive celiac genetics were prospectively included. Fifty had villous atrophy, 70 lymphocytic enteritis, and 85 normal histology. Eight patients with non-celiac atrophy and 15 with lymphocytic enteritis secondary to Helicobacter pylori acted as control group. Duodenal biopsies were obtained to assess both CD IEL flow cytometric (complete or incomplete and IF patterns. RESULTS: Sensitivity of IF, and complete and incomplete cytometric patterns for CD diagnosis in patients with positive serology (Marsh 1+3 was 92%, 85 and 97% respectively, but only the complete cytometric pattern had 100% specificity. Twelve seropositive and 8 seronegative Marsh 1 patients had a CD diagnosis at inclusion or after gluten free-diet, respectively. CD cytometric pattern showed a better diagnostic performance than both IF pattern and serology for CD diagnosis in lymphocytic enteritis at baseline (95% vs 60% vs 60%, p = 0.039. CONCLUSIONS: Analysis of the IEL flow cytometric pattern is a fast, accurate method for identifying CD in the initial diagnostic biopsy of patients presenting with lymphocytic enteritis, even in seronegative patients, and seems to be better than anti-TG2 intestinal deposits.

  9. Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

    Science.gov (United States)

    Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

    2015-10-06

    Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

  10. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    Science.gov (United States)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  11. Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

    Directory of Open Access Journals (Sweden)

    Braz Lúcia M.A.

    1996-01-01

    Full Text Available Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

  12. Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing.

    Science.gov (United States)

    Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J; Katayev, Alexander; Fleming, James K

    2018-01-01

    Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6-97.5% of patterns and 71.0-93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

  13. Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

    Directory of Open Access Journals (Sweden)

    Vincent Ricchiuti

    2018-05-01

    Full Text Available Indirect immunofluorescence (IIF is considered by the American College of Rheumatology (ACR and the international consensus on ANA patterns (ICAP the gold standard for the screening of anti-nuclear antibodies (ANA. As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6% than when compared to the current method (89.4%. Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

  14. Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

    Directory of Open Access Journals (Sweden)

    Shinnosuke Inoue

    Full Text Available An occupationally safe (biosafe sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

  15. Spatial Mixture Modelling for Unobserved Point Processes: Examples in Immunofluorescence Histology.

    Science.gov (United States)

    Ji, Chunlin; Merl, Daniel; Kepler, Thomas B; West, Mike

    2009-12-04

    We discuss Bayesian modelling and computational methods in analysis of indirectly observed spatial point processes. The context involves noisy measurements on an underlying point process that provide indirect and noisy data on locations of point outcomes. We are interested in problems in which the spatial intensity function may be highly heterogenous, and so is modelled via flexible nonparametric Bayesian mixture models. Analysis aims to estimate the underlying intensity function and the abundance of realized but unobserved points. Our motivating applications involve immunological studies of multiple fluorescent intensity images in sections of lymphatic tissue where the point processes represent geographical configurations of cells. We are interested in estimating intensity functions and cell abundance for each of a series of such data sets to facilitate comparisons of outcomes at different times and with respect to differing experimental conditions. The analysis is heavily computational, utilizing recently introduced MCMC approaches for spatial point process mixtures and extending them to the broader new context here of unobserved outcomes. Further, our example applications are problems in which the individual objects of interest are not simply points, but rather small groups of pixels; this implies a need to work at an aggregate pixel region level and we develop the resulting novel methodology for this. Two examples with with immunofluorescence histology data demonstrate the models and computational methodology.

  16. Pattern of glomerular diseases in oman: A study based on light microscopy and immunofluorescence

    Directory of Open Access Journals (Sweden)

    Nasar Yousuf Alwahaibi

    2013-01-01

    Full Text Available Light microscopy and immunofluorescence play an important part in the final diagnosis of renal biopsy. The aim of this study was to analyze the pattern of various glomerular diseases in Oman. A total of 424 renal biopsies were retrospectively analyzed at the Sultan Qaboos University Hospital between 1999 and 2010. Focal and segmental glomerulosclerosis (FSGS, minimal change disease (MCD, membranous glomerulopathy (MGN and IgA nephropathy were the most common primary glomerular diseases encountered, accounting for 21.2%, 17%, 12.3% and 8.3%, respectively, of all cases. Lupus nephritis was the most common secondary glomerular disease and was the most prevalent among all biopsies, accounting for 30.4% of all biopsies. Amyloidosis was seen in only two cases. The presence of fluorescein isothiocyanatefibrin in all renal cases was low when compared with IgG, IgA, IgM, C3 and C1q markers. In conclusion, based on the findings of this study, lupus nephritis was the most common of all glomerular diseases and FSGS was the most common primary glomerular disease. The importance of fluorescein isothiocyanate-fibrin in the diagnosis of renal biopsy needs to be further investigated.

  17. Periodic Granulocyte Count Measuring Is Useful for Detecting Asymptomatic Agranulocytosis in Antithyroid Drug-Treated Patients with Graves' Disease.

    Science.gov (United States)

    Nakamura, Hirotoshi; Ide, Akane; Kudo, Takumi; Nishihara, Eijun; Ito, Mitsuru; Miyauchi, Akira

    2016-12-01

    Finding agranulocytosis (AG) at an early stage is important to improve outcome, but periodic granulocyte count monitoring is not generally recommended for patients with Graves' disease, because AG develops suddenly. At the Kuma Hospital, Graves' patients under antithyroid drug (ATD) treatment in an outpatient clinic have a granulocyte count examination during each visit, and if it is Graves' disease were 131 I-radioisotope therapy (19 patients), thyroidectomy (2 patients), inorganic iodine (1 patient), or another ATD (1 patient). Among the 33 GP patients, 31 (94%), including 20 asymptomatic cases, were discovered during periodic granulocyte count monitoring. Most of them stopped ATD, and other treatments for Graves' disease were selected. Periodic monitoring of granulocyte counts is useful for identifying AG and GP patients with no or minimum infection symptoms.

  18. Bone infection in patients suspected of complicating osteomyelitis: the diagnostic value of dual isotope bone-granulocyte scintigraphy

    DEFF Research Database (Denmark)

    Buhl, Thora; Stentzer, Kim; Hede, Adam

    2005-01-01

    : Simultaneous dual isotope bone-granulocyte scintigraphic images were obtained in 42 consecutive patients in whom conventional X-ray, erythrocyte sedimentation rate, and C-reactive protein were also available. 99mTc MDP bone and 111In labelled granulocyte imaging was obtained simultaneously. The images were...... interpreted as positive for osteomyelitis if regions of interests of pathologic 111In granulocyte accumulation included 99mTc MDP activity on the bone images (except in the spine). RESULTS: The sensitivity, specificity, and accuracy were 84, 71 and 79%, respectively, for simultaneous, dual isotope bone......AIM: The purpose of this study was to evaluate the diagnostic value of dual isotope bone-granulocyte scintigraphy in patients with known bone pathology clinically suspected of osteomyelitis, i.e. complicating osteomyelitis, using per-operative bacterial culture from bone as reference. METHODS...

  19. Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy

    NARCIS (Netherlands)

    Jansen, K; Fabro, C; Artero, C; Feuilloley, M; Vaudry, H; Fasolo, A; Franzoni, MF

    Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and

  20. Immunofluorescence-based screening identifies germ cell associated microRNA 302 as an antagonist to p63 expression

    DEFF Research Database (Denmark)

    Scheel, Andreas Hans Joachim; Beyer, Ulrike; Agami, Reuven

    2009-01-01

    The tumor suppressor homologue p63 is required for proper skin and limb development, but specific isoforms of it also act as a "guardian of the germline." To gain insight into the regulation of p63 expression, we performed immunofluorescence-based screening assays. Using a large collection of micro...

  1. Polycythemia vera treated with /sup 32/P and myleran: Development of chronic granulocytic leukemia with chromosomal abnormalities in one patient

    Energy Technology Data Exchange (ETDEWEB)

    Stavem, P; Sandnes, K [Rikshospitalet, Oslo (Norway); Hagen, C.B. van der [Oslo Univ. (Norway); Vogt, E [Statens Institutt for Folkehelse, Oslo, Norway

    1975-01-01

    Chronic granulocytic leukemia developed in a 59-year-old woman who had previously received a total of 21 mCl /sup 32/P for polycythemia vera. She was treated with Myleran (busulphan) for her chronic granulocytic leukemia. Cytogenetic studies revealed deletion of chromosomes No. 8 and 12, and translocation between 1 and 8. The patient also developed a severe autoimmune hemolytic anemia, for which she received prednisone treatment. She died with a perforated stomach ulcer. (INIS)

  2. Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

    Science.gov (United States)

    Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

    2016-11-01

    Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G 0 /G 1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family

  3. Use of indium-111-oxinate-labelled granulocytes and thrombocytes in kidney transplantation

    International Nuclear Information System (INIS)

    Royen, E.A. van; Schoot, J.B. van der; Hardeman, M.R.; Surachno, S.; Veen, J.H. ten; Vreeken, J.; Wilmink, J.M.

    1981-01-01

    The diagnostic use of 111 In-oxinate-labelled granulocytes and thrombocytes in kidney graft rejection was studied in 39 transplant patients. Normal values were established for the deposition of these cells in stable, functioning kidney grafts. Although some 111 In granulocyte accumulation occurred in the graft during rejection, the increase was too slight to render the method suitable for the early diagnosis of rejection. Significant increased 111 In thrombocyte deposition was found during rejection periods, although large differences were observed in the degree of accumulation. Severity or type of rejection may relate to these differences. Post-transplantation follow-up by 111 In thrombocyte scintigraphy did not result in a much earlier diagnosis of rejection than classic clinical signs. However, more frequent bedside activity determinations might do so. (author)

  4. Efficacy and safety of granulocyte, monocyte/macrophage adsorptive in pediatric ulcerative colitis

    DEFF Research Database (Denmark)

    Ruuska, Tarja; Küster, Peter; Grahnquist, Lena

    2016-01-01

    AIM: To investigate efficacy and safety for granulocyte, monocyte apheresis in a population of pediatric patients with ulcerative colitis. METHODS: The ADAPT study was a prospective, open-label, multicenter study in pediatric patients with moderate, active ulcerative colitis with pediatric...... ulcerative colitis activity index (PUCAI) of 35-64. Patients received one weekly apheresis with Adacolumn(®) granulocyte, monocyte/macrophage adsorptive (GMA) apheresis over 5 consecutive weeks, optionally followed by up to 3 additional apheresis treatments over 3 consecutive weeks. The primary endpoint...... mg daily on average from Baseline to week 12. CONCLUSION: Adacolumn(®) GMA apheresis treatment was effective in pediatric patients with moderate active Ulcerative Colitis. No new safety signals were reported. The present data contribute to considering GMA apheresis as a therapeutic option...

  5. Granulocyte colony-stimulating factor protects mice during respiratory virus infections.

    Directory of Open Access Journals (Sweden)

    Tamar Hermesh

    Full Text Available A burst in the production of pro-inflammatory molecules characterizes the beginning of the host response to infection. Cytokines, chemokines, and growth factors work in concert to control pathogen replication and activate innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF mobilizes and activates hematopoietic cells from the bone marrow, and it has been shown to mediate the generation of effective immunity against bacterial and fungal infections. G-CSF is produced at high levels in the lungs during infection with influenza and parainfluenza viruses, but its role during these infections is unknown. Here we show that during infection of mice with a non-lethal dose of influenza or Sendai virus, G-CSF promotes the accumulation of activated Ly6G+ granulocytes that control the extent of the lung pro-inflammatory response. Remarkably, these G-CSF-mediated effects facilitate viral clearance and sustain mouse survival.

  6. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    Science.gov (United States)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  7. Increased Oxidative Stress Response in Granulocytes from Older Patients with a Hip Fracture May Account for Slow Regeneration

    Directory of Open Access Journals (Sweden)

    Zhiyong Wang

    2014-01-01

    Full Text Available Proximal femur fracture, a typical fracture of the elderly, is often associated with morbidity, reduced quality of life, impaired physical function and increased mortality. There exists evidence that responses of the hematopoietic microenvironment to fractures change with age. Therefore, we investigated oxidative stress markers and oxidative stress-related MAPK activation in granulocytes from the young and the elderly with and without fractured long bones. Lipid peroxidation levels were increased in the elderly controls and patients. Aged granulocytes were more sensitive towards oxidative stress induced damage than young granulocytes. This might be due to the basally increased expression of SOD-1 in the elderly, which was not further induced by fractures, as observed in young patients. This might be caused by an altered MAPK activation. In aged granulocytes basal p38 and JNK activities were increased and basal ERK1/2 activity was decreased. Following fracture, JNK activity decreased, while ERK1/2 and p38 activities increased in both age groups. Control experiments with HL60 cells revealed that the observed p38 activation depends strongly on age. Summarizing, we observed age-dependent changes in the oxidative stress response system of granulocytes after fractures, for example, altered MAPK activation and SOD-1 expression. This makes aged granulocytes vulnerable to the stress stimuli of the fracture and following surgery.

  8. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    International Nuclear Information System (INIS)

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H.

    1990-01-01

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis

  9. Micronuclei frequency in circulating erythrocytes from rainbow trout (Oncorhynchus mykiss) subjected to radiation, an image analysis and flow cytometric study

    International Nuclear Information System (INIS)

    Schultz, N.; Norrgren, L.; Grawe, J.; Johannisson, A.; Medhage, O.

    1993-01-01

    Rainbow trout (oncorhynchus mykiss) were exposed to a single X-ray dose of 4 Gy. The frequency of micronuclei in the peripheral erythrocytes was investigated at regular intervals up to 58 days after the exposure. A flow cytometric method and a semi-automatic image analysis method were used to estimate the micronuclei frequency. The results show that both methods can detect an increased frequency of micronuclei in peripheral erythrocytes from exposed fish. However, the semi-automatic image analysis method was the most stable and sensitive. (Author)

  10. Human Granulocyte Colony-Stimulating Factor (hG-CSF) Expression in Plastids of Lactuca sativa

    OpenAIRE

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Background: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. Methods: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA ter...

  11. ITAM-like signalling for efficient phagocytosis : The paradigm of the granulocyte receptor CEACAM3

    OpenAIRE

    Pils, Stefan

    2010-01-01

    Human CEACAM3 is a tailor-made receptor of the innate immune system to fight pathogens exploiting epithelial CEACAM-family members for colonisation and invasion of their host. Previous studies established CEACAM3 as the receptor facilitating rapid phagocytosis and elimination of N. gonorrhoeae by human granulocytes. The studies reported here set out to shed light on the evolution of this highly specialised receptor and the associated signalling machinery.CEACAM3 arose from exon shuffling afte...

  12. Assessment of the Total Inflammatory Potential of Bioaerosols by Using a Granulocyte Assay▿

    OpenAIRE

    Timm, Michael; Madsen, Anne Mette; Hansen, Jørgen Vinsløv; Moesby, Lise; Hansen, Erik Wind

    2009-01-01

    Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by tr...

  13. A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

    Directory of Open Access Journals (Sweden)

    Abraham A

    2009-01-01

    Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

  14. Determination of cutoff of ELISA and immunofluorescence assay for scrub typhus

    Directory of Open Access Journals (Sweden)

    Nitin Gupta

    2016-01-01

    Full Text Available Background: The most common method employed for diagnosis of scrub typhus is serology. It is widely known that demonstration of ≥4-fold rise in titers of antibody in paired sera is required for diagnosis. However, for guidance of initial treatment, there is a need for rapid diagnosis at the time of admission. Therefore, there is a need for standardized region specific cutoff titers at the time of admission. Materials and Methods: A total of 258 patients of all age groups with clinically suspected scrub typhus over a period of 24 months (October 2013-October 2015 were enrolled. Serum samples of these patients were subjected to immunofluorescent antibody (IFA for immunoglobulin M (IgM (Fuller Labs, USA with dilutions of 1:64, 1:128, 1:256, and 1:512. Serum samples of all 258 patients were subjected to IgM ELISA (Inbios Inc., USA. Any patient with response to antibiotics within 48 h accompanied by either presence of an eschar or positivity by polymerase chain reaction was taken as positive. Receiver operating characteristic (ROC curve was drawn to generate cutoff for these tests. Results: A total of 20 patients were diagnosed as cases of scrub typhus. The ROC curve analysis revealed a cutoff optical density value of 0.87 with sensitivity and specificity of 100% and 94.12%, respectively. ROC curve analysis of IFA revealed sensitivity and specificity of 100% and 93.5%, respectively at 1:64 dilution. Conclusion: Considering cost constraints, centers in and around New Delhi region can use the cutoffs we determined for the diagnosis of scrub typhus.

  15. Neuromyelitis optica immunoglobulin G in Chinese patients detected by immunofluorescence assay on a monkey brain substrate.

    Science.gov (United States)

    Long, Youming; Hu, Xueqiang; Peng, Fuhua; Lu, Zhengqi; Wang, Yuge; Yang, Yu; Qiu, Wei

    2012-01-01

    Serum neuromyelitis optica immunoglobulin G (NMO-IgG) is used as a biomarker to differentiate between neuromyelitis optica (NMO) and multiple sclerosis (MS). However, the original assay is expensive and complex and shows low sensitivity. Here, we investigated the potential of NMO-IgG detection using an indirect immunofluorescence (IIF) assay on monkey brains. NMO-IgG seroprevalence was determined in 168 samples by an IIF assay on a monkey brain substrate. The data were compared with those from a standard mouse brain IIF assay using McNemar and kappa tests. Thirty-one of 50 (62%) NMO patients, 7 of 18 (38.9%) longitudinally extensive transverse myelitis patients, 6 of 57 (10.5%) MS patients, and 5 of 10 (50%) optic neuritis patients were seropositive for NMO-IgG. None of the acute partial transverse myelitis patients (n = 3) or healthy controls (n = 20) was positive. Thus, the sensitivity of the test was 62% for the patients with clinically definite NMO. The specificity was 89.5%, considering the 57 MS patients as the control group. The modified IIF assay on monkey brains and the standard IIF assay based on mouse brains were not significantly different (McNemar test; p = 1.000). The two assays were concordant in 39 seropositive samples and 100 seronegative samples (kappa test; kappa = 0.592, p monkey brain assay was no better than the standard mouse brain IIF assay, we affirmed that NMO-IgG is a sensitive and specific biomarker to differentiate between NMO and MS. Copyright © 2011 S. Karger AG, Basel.

  16. Granulocytes and vascularization regulate uterine bleeding and tissue remodeling in a mouse menstruation model.

    Directory of Open Access Journals (Sweden)

    Astrid Menning

    Full Text Available Menstruation-associated disorders negatively interfere with the quality of life of many women. However, mechanisms underlying pathogenesis of menstrual disorders remain poorly investigated up to date. Among others, this is based on a lack of appropriate pre-clinical animal models. We here employ a mouse menstruation model induced by priming mice with gonadal hormones and application of a physical stimulus into the uterus followed by progesterone removal. As in women, these events are accompanied by menstrual-like bleeding and tissue remodeling processes, i.e. disintegration of decidualized endometrium, as well as subsequent repair. We demonstrate that the onset of bleeding coincides with strong upregulation of inflammatory mediators and massive granulocyte influx into the uterus. Uterine granulocytes play a central role in regulating local tissue remodeling since depletion of these cells results in dysregulated expression of matrix modifying enzymes. As described here for the first time, uterine blood loss can be quantified by help of tampon-like cotton pads. Using this novel technique, we reveal that blood loss is strongly reduced upon inhibition of endometrial vascularization and thus, is a key regulator of menstrual bleeding. Taken together, we here identify angiogenesis and infiltrating granulocytes as critical determinants of uterine bleeding and tissue remodeling in a mouse menstruation model. Importantly, our study provides a technical and scientific basis allowing quantification of uterine blood loss in mice and thus, assessment of therapeutic intervention, proving great potential for future use in basic research and drug discovery.

  17. Immature granulocyte detection by the SE-9000 haematology analyser during pregnancy.

    Science.gov (United States)

    Fernández-Suárez, A; Pascual, V T; Gimenez, M T F; Hernández, J F S

    2003-12-01

    The objective of this study was to determine the nature of the alarm for immature granulocytes appearing in haemograms from pregnant women, as detected by the immature cell information channel (IMI) of the SE-9000 automated haematology analyser. Of all tests run on pregnant women in a 4-month period (n = 698), the first 100 haemograms with immature granulocyte alarms (14.33%) were collected. Each of these samples was then stained with Wright-Giemsa stain. The following variables were also analysed: age of the mother, trimester and days of gestation, type of delivery, weight and sex of the baby, and Apgar score. Most pregnant women were in the third trimester of gestation (82%) when an alarm was noted on the IMI channel. Of the patients, 62% had normal deliveries. The most frequent complication was obstructed delivery (23%). Mean percentages by microscopic counts of band cells, metamyelocytes, and myelocytes were 2.99, 0.45, and 0.19%, respectively. There was a statistically significant correlation for all cell types between the SE-9000 and the manual count method. No association was observed between the presence of immature granulocytes and the clinical variables analysed. The SE-9000 analyser shows high sensitivity in the IMI channel for detection of immature forms.

  18. Radionuclides and inflammatory bowel diseases: 99mTc-sucralfate and 111 In-tropolonate-granulocytes

    International Nuclear Information System (INIS)

    Deveaux, M.; Macaigne, O.; Lecouffe, P.; Hossein-Foucher, CL.; Meuriot, S.; Venel, H.; Cortot, A.; Marchandise, X.

    1989-01-01

    111 In-tropolonate-granulocytes (G- 111 In) study was performed in 16 patients with inflammatory bowel disease (IBD). Images were obtained 3 h and 20 h post-injection. Counting of four days feacal excretion was more accurately expressed as daily intestinal granulocytes clearance. The day before, 12 of the patients had a sucralfate- 99m Tc scan (S- 99m Tc). Correlation between radioendoscopic data and S- 99m Tc scans was poor in 9 instances and there was no correlation between scan activity index and clinical and biological assessment. Correlation between G- 111 In scans and radioendoscopic data was excellent in 13 instances. Scan activity index was correlated with the Best index and with the intestinal alpha-1-antitrypsine clearance. Faecal excretion (range. 1 - 40%) and daily intestinal granulocytes clearance values (range. 03 - 50 milliards) were only correlated with protein loss parameters. So S- 99m Tc scan does not appear to be reliable, while intestinal G- 111 In clearance, not such easy to perform, can give an accurate assessment of IBD activity [fr

  19. Effects of humoral factors on amplification of nonrecognizable erythrocytic and granulocytic precursors

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Carsten, A.L.; Cohen, R.; Miller, M.E.; Moccia, G.

    1978-01-01

    The purpose of these studies was to evaluate the effects of humoral factors on amplification of nonrecognizable erythrocytic and granulocytic precursors using the in vivo plasma clot diffusion chamber and the in vitro plasma clot culture methods. Plasma erythropoietin levels changes in the reticulocyte concentration and hematocrits of irradiated and non-irradiated Long-Evans rats exposed to hypoxia were also determined. While erythropoietin plasma levels appeared to effect BFU-E and CFU-E growth, results suggest erythropoietin may not be the sole regulator of red cell production and that inhibitors or chalone-like mechanisms may be involved. Measurements made on granulocyte precursors treated with CSF containing L-cell conditioned medium revealed granulocytic colonies and burst-like formations, similar to those seen for erythrocytic growth. There is strong evidence suggesting that CSF is a regulator of granulopoiesis; however, it is not the sole regulator and it appears that inhibitors may play an in vivo role. Growth of colonies with cell numbers not a power of 2 implies either asymmetric nitosis due to loss of genetic information required for continuing division, or differences in concentration of, or ability to recognize inhibitory factors. These possibilities are examined on the basis of results using in vivo and in vitro culture techniques

  20. Increased granulocytic, erythrocytic, and megakaryocytic progenitors in myelofibrosis with myeloid metaplasia

    International Nuclear Information System (INIS)

    Chikkappa, G.; Carsten, A.L.; Chanana, A.D.; Chandra, P.; Cronkite, E.P.

    1978-01-01

    Nucleated cells obtained from blood and/or bone marrow of patients with myelofibrosis with myeloid metaplasia (MMM) were cultured in diffusion chambers (DC) implanted into the peritoneal cavities of irradiated mice. A total of five blood studies and two bone marrow studies were performed using cells obtained from five patients. The DC were harvested at intervals from the host mice and the total and differential cellularity of DC contents were evaluated. The results obtained from MMM cultures were compared with those from similar cultures of blood cells and marrow cells of four and six normal individuals respectively. The proliferation and maturation of the granulocytic, erythrocytic, and megakaryocytic lines in MMM cultures occurred in an orderly fashion as they occur in vivo. The patterns of proliferation and maturation of the three cell lines in cultures after day 7 suggest that they primarily originate from progenitor cells. The numbers of granulocytes in the multiplicative pool, recognizable red cell precursors, and megakaryocytes recovered were significantly greater from the MMM cultures than those from the normal blood or marrow cultures. These results suggest that the blood and marrow cells of MMM patients have increased numbers of progenitors for granulocytes, erythrocytes and megakaryocytes

  1. Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs.

    Science.gov (United States)

    Cremer, Signe E; Krogh, Anne K H; Hedström, Matilda E K; Christiansen, Liselotte B; Tarnow, Inge; Kristensen, Annemarie T

    2018-06-01

    Platelet microparticles (PMPs) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. Microparticles (MP) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Anti-CD61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD61 + /AnV - concentrations were analyzed with/without a calcium buffer. CD61 + /AnV - , CD61 + /AnV + , and CD61 - /AnV + MP quantification were validated in 10 healthy dogs. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses and detection limits (DLs) were calculated. CD61 + /AnV - concentrations were higher in calcium buffer; 841,800 MP/μL (526,000-1,666,200) vs without; 474,200 MP/μL (278,800-997,500), P < .05. In WB, PMP were above DLs and demonstrated acceptable (<20%) intra-assay and inter-assay CVs in 9/10 dogs: 1.7% (0.5-8.9) and 9.0% (0.9-11.9), respectively, for CD61 + /AnV - and 2.4% (0.2-8.7) and 7.8% (0.0-12.8), respectively, for CD61 + /AnV + . Acceptable CVs were not seen for the CD61 - /AnV + MP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs. Calcium induced higher in vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference. © 2018 American Society for Veterinary Clinical Pathology.

  2. Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

    Science.gov (United States)

    Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

    1996-03-01

    A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

  3. Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

    Science.gov (United States)

    Carson, Christine F; Inglis, Timothy JJ

    2018-01-01

    This study investigated aerosolized viable bacteria in a university research laboratory during operation of an acoustic-assisted flow cytometer for antimicrobial susceptibility testing by sampling room air before, during and after flow cytometer use. The aim was to assess the risk associated with use of an acoustic-assisted flow cytometer analyzing unfixed bacterial suspensions. Air sampling in a nearby clinical laboratory was conducted during the same period to provide context for the existing background of microorganisms that would be detected in the air. The three species of bacteria undergoing analysis by flow cytometer in the research laboratory were Klebsiella pneumoniae, Burkholderia thailandensis and Streptococcus pneumoniae. None of these was detected from multiple 1000 L air samples acquired in the research laboratory environment. The main cultured bacteria in both locations were skin commensal and environmental bacteria, presumed to have been disturbed or dispersed in laboratory air by personnel movements during routine laboratory activities. The concentrations of bacteria detected in research laboratory air samples were reduced after interventional cleaning measures were introduced and were lower than those in the diagnostic clinical microbiology laboratory. We conclude that our flow cytometric analyses of unfixed suspensions of K. pneumoniae, B. thailandensis and S. pneumoniae do not pose a risk to cytometer operators or other personnel in the laboratory but caution against extrapolation of our results to other bacteria and/or different flow cytometric experimental procedures. PMID:29608197

  4. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.

  5. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    International Nuclear Information System (INIS)

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats

  6. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases.

    Science.gov (United States)

    Sano, Susana Mariela; Quarracino, María Cecilia; Aguas, Silvia Cristina; González, Ernestina Jesús; Harada, Laura; Krupitzki, Hugo; Mordoh, Ana

    2008-05-01

    Direct immunofluorescence (DIF) is widely used for the diagnosis of bullous diseases and other autoimmune pathologies such as oral lichen planus. There is no evidence in the literature on how the following variants influence the detection rate of DIF: intraoral site chosen for the biopsy, perilesional locus or distant site from the clinical lesion, number of biopsies and instrument used. to determine if the following variants influenced the sensitivity (detection rate): intraoral site chosen for the biopsy, perilesional or distant site from the clinical lesion, number of biopsies and instrument used (punch or scalpel). A retrospective study was done at the Cátedra de Patología y Clínica Bucodental II at the Facultad de Odontología, Universidad de Buenos Aires; 136 clinical medical histories were revised for the period March 2000 - March 2005 corresponding to patients with clinical diagnosis of OLP and bullous diseases (vulgar pemphigus, bullous pemphigoid and cicatricial pemphigoid). DIF detection rate was 65.8% in patients with OLP, 66.7% in cicatricial pemphigoid patients, in bullous pemphigoid 55.6%, in pemphigus vulgaris 100%, and in those cases in which certain diagnosis could not be obtained, the DIF positivity rate was 45.5% (Pearson chi(2) (4)= 21.5398 Pr= 0.000). There was no statistically significant difference between the different sites of biopsy (Fisher exact test: 0.825). DIF detection rate in perilesional biopsies was 66.1% and in those distant from the site of clinical lesion was 64.7% (Pearson chi(2) v1)= 0.0073 Pr= 0.932. When the number of biopsies were incremented, DIF detection rate also incremented (Pearson chi(2) = 8.7247 Pr= 0.003). The biopsies taken with punch had a higher detection rate than those taken with scalpel (39.1% versus 71.7%) (Pearson chi(2) = 49.0522 Pr= 0.000). While not statistically significant, the tendency outlined in this study indicates there are intraoral regions in which the detection rate of the DIF technique is

  7. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-κB translocation

    International Nuclear Information System (INIS)

    Jawan, Bruno; Kao, Y.-H.; Goto, Shigeru; Pan, M.-C.; Lin, Y.-C.; Hsu, L.-W.; Nakano, Toshiaki; Lai, C.-Y.; Sun, C.-K.; Cheng, Y.-F.; Tai, M.-H.

    2008-01-01

    Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 μM after 48 h incubation. Pretreatment with 100 μM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IκBα, as well as the nuclear translocation of NF-κB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-κB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers

  8. Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

    International Nuclear Information System (INIS)

    Fiebach, H.; Uckert, W.; Micheel, B.

    1982-01-01

    Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle. (author)

  9. Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

    Energy Technology Data Exchange (ETDEWEB)

    Fiebach, H.; Uckert, W.; Micheel, B. (Akademie der Wissenschaften der DDR, Berlin. Zentralinstitut fuer Krebsforschung)

    Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle.

  10. Application of indirect immunofluorescent test with an improved HEp-2 substrate tranfected with human Ro60/SSA autoantigens

    International Nuclear Information System (INIS)

    Lv Liangjing; Chen Shunle; Gu Yueying; Shen Nan; Bao Chunde; Wang Yuan; Xue Feng; Ye Peng; Yu Chongzhao

    2006-01-01

    To develop an improved substrate for indirect immunofluorescent test (IIF) to detect anti-Ro/SSA autoantibodies, the human 60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from placental cDNA library using PCR and were cloned into the mammalian expression vectorpEGFP-C1. Then the recombinant plasmids were transfected into HEp-2 cells. We con- firmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of fluorescence microscopy, immunoblotting and IIF. HEp-2 and HEp-Ro60 was analyzed by IIF using a panel of 10 precipitinpositive anti-Ro human sera simultaneously. Stable expression of Ro60-GFP (green fluorescent protein) fusion proteins maintained ten more generations. And Ro60-GFP kept the antigenicity of Ro and had its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer, P<0.01). Eight (8/10) positive an- ti-Ro sera showed characteristic immunofluorescent pattern on HEp-Ro60, including two sera which were antinuclear antibodies (ANA) negative on untransfected HEp-2. IIF-ANA in all healthy sera were negative on HEp-Ro60. As a kind of new substrate of IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells kept the immunofluorescent property of HEp-2 cells in IIF-ANA tests and could be employed as substrate for the routine IIF-ANA detection. The method improved the sensitivity of IIF-ANA. (authors)

  11. Flow cytometric analysis of lymphocytes and lymphocyte subpopulations in induced sputum from patients with asthma

    Directory of Open Access Journals (Sweden)

    Yutaro Shiota

    2000-01-01

    Full Text Available Study objectives were to compare the numbers of lymphocytes and lymphocyte subpopulations in induced sputum from asthmatic patients and from healthy subjects, and to determine the effect of inhaled anti-asthmatic steroid therapy on these cell numbers. Hypertonic saline inhalation was used to non-invasively induce sputum samples in 34 patients with bronchial asthma and 21 healthy subjects. The sputum samples were reduced with dithioerythritol and absolute numbers of lymphocytes and lymphocyte subpopulations were assessed by direct immunofluorescence and flow cytometry. To assess the effect of beclomethasone dipropionate (BDP on induced sputum, numbers of lymphocytes and lymphocyte subpopulations in sputum also were evaluated after 4 weeks of BDP inhalation treatment in seven asthmatic patients. An adequate sample was obtained in 85.3% of patients with asthma and in 79.2% of the healthy subjects. Induced sputum from patients with asthma had increased numbers of lymphocytes (P = 0.009; CD4+ cells (P = 0.044; CD4+ cells-bearing interleukin-2 receptor (CD25; P = 0.016; and CD4+ cells bearing human histocompatibility leukocyte antigen (HLA-DR (P = 0.033. CD8+ cells were not increased in asthmatic patients. In patients treated with inhaled steroids, numbers of lymphocytes, CD4+ cells, CD25-bearing CD4+ cells and HLA-DR-bearing CD4+ cells in sputum decreased from pretreatment numbers (P = 0.016, 0.002, 0.003 and 0.002, respectively. Analysis of lymphocytes in induced sputum by flow cytometry is useful in assessing bronchial inflammation, and activated CD4+ lymphocytes may play a key role in the pathogenesis of airway inflammation in bronchial asthma.

  12. CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

    1993-01-15

    In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

  13. Hematological importance of pseudoeosinophilic granulocytes in acclimation of common carp (Cyprinus carpio Linnaeus, 1758

    Directory of Open Access Journals (Sweden)

    Damir Suljevic

    2017-03-01

    Full Text Available Adaptation mechanisms as response to water content, oxygen level and pollutants are very important and they can be interpreted by hematological analysis. The aim of this study was the analysis of hematological and immune adaptations of common carp (Cyprinus carpio Linnaeus, 1758 to thermal stress. All specimens were divided into a control and experimental group. The control group of fish was exposed to a constant water temperature of 10°C. We induced thermal stress in experimental fish by gradually heating water to 28°C, held for 30 minutes and then comparing the obtained results with the control fish. Short-term hyperthermia lead to an increase of the number of leukocytes, especially pseudoeosinophilic granulocytes and monocytes, while the number of neutrophils and lymphocytes was reduced. The analysis of the leukocyte number and differential blood count in the control group showed high individual variation of segmented granulocytes, monocytes and pseudoeosinophilic granulocytes. Statistically significant differences (p=0.00 were found for the white blood cells, nonsegmented neutrophils and pseudoeosinophils between the control and experimental group. The experimental group of males had an increased number of white blood cells, monocytes and pseudoeosinophils, where significant differences were found for nonsegmented and total neutrophils and also for pseudoeosinophils (p=0.00, lymphocytes (p=0.01 and monocytes (p=0.03. Females had an increased total number of white blood cells, lymphocytes, monocytes and pseudoeosinophils, while significant differences (p=0.00 were obtained in the number of white blood cells, non segmented and total neutrophils and pseudoeosinophils between the control and experimental group. Adaptation mechanisms in carp after water temperature heating are mostly reflected in the increase of pseudoeosinophils and the decrease of neutrophils.

  14. Antibodies to granulocytic ehrlichiae in white-footed and cotton mice in eastern United States.

    Science.gov (United States)

    Magnarelli, L A; Stafford, K C; Ijdo, J W; Fikrig, E; Oliver, J H; Hutcheson, H J; Boone, J L

    1999-04-01

    Serum samples, collected from Peromyscus leucopus (white-footed mouse) or Peromyscus gossypinus (cotton mouse) during 1987 through 1990 in Florida, Georgia, Maryland, Mississippi, and North Carolina (USA), and in 1997 in southern Connecticut were analyzed by indirect fluorescent antibody (IFA) staining methods or Western blot procedures for antibodies to granulocytic ehrlichiae. Of the 82 sera from white-footed mice in Connecticut tested by IFA methods with either the BDS or NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent, 45 (55%) and 42 (51%) of the samples contained antibodies to these strains, respectively, at concentrations ranging from 1:80 to 1:2560. One (2%) of 43 sera from P. leucopus captured in Assateague Island (Maryland) had a titer of 1:80, while three (20%) of 15 sera from P. gossypinus, captured in Sapelo Island (Georgia) and four (40%) of 10 sera from cotton mice caught in Amelia Island (Florida) had antibodies to the NCH-1 strain at titers of 1:160 to 1:1,280. Fifty-five sera from P. leucopus in Cape Hatteras (North Carolina) and 30 sera from P. gossypinus in Mississippi were negative. Western blot analyses confirmed seropositivity for 19 (95%) of 20 mouse sera positive by IFA staining methods, including samples from both mouse species captured in Connecticut, Maryland, or Florida. There were key banding patterns to proteins having molecular masses of about 44, 80, 105, 110, or 120 kDa. Both serologic assays can be used to determine if mice have been exposed to granulocytic ehrlichiae. These rodents also may be useful in surveillance programs to identify endemic sites for HGE and in performing laboratory studies on immune responses to the etiologic agent.

  15. Cytotoxic immigration of granulocytes into megakaryocytes as a late consequence of irradiation

    International Nuclear Information System (INIS)

    Calvo, W.; Alabi, R.; Nothdurft, W.; Fliedner, T.M.

    1994-01-01

    The immigration of neutrophilic granulocytes into megakaryocyte was studied in the bone marrow of normal and X-irradiated beagle under various exposure conditions. Two groups of dogs received homogeneous total-body irradiation. One group received a dose of 1.6 Gy and the other received a dose of 2.4 Gy (midline tissue). A third group was irradiated from the left side of the body only. This exposure resulted in an inhomogeneous total-body irradiation (entrance dose 3.8 Gy, exit dose 0.9 Gy). A fourth group of animals received partial-body irradiation with a dose of 11.7 Gy delivered to the anterior two-thirds of the body, thereby subjecting about 70% of the hemopoietic marrow to irradiation. Dogs of a fifth group remained unexposed to irradiation and served as controls. The marrow was analyzed in sections of the ribs approximately 1 year after irradiation. The total number of megakaryocytes in one section was evaluated. The number of megakaryocytes showing granulocytes in their cytoplasm was determined and expressed as a percentage. This phenomenon can be explained as cytotoxic immigration of granulocytes into megakaryocytes. It was observed in approximately 1-2 of the megakaryocytes in the marrow of normal dogs. One year after irradiation the value increased of normal dogs. One year after irradiation the value increased to 10-26%. It was observed that neutrophilic granuloytes penetrated only into the large mature megakaryocytes in which the nuclei were most pyknotic. This phenomenon may be considered as a late effect of irradiation. 15 refs., 4 figs., 2 tabs

  16. Recurrent spleen enlargement during cyclic granulocyte-macrophage colony-stimulating factor therapy for myelodysplastic syndrome

    International Nuclear Information System (INIS)

    Delmer, A.; Karmochkine, M.; Cadiou, M.; Gerhartz, H.; Zittoun, R.

    1990-01-01

    A 65-year-old woman with refractory anemia with excess of blasts received sequential courses of granulocyte-macrophage colony-stimulating factor therapy (GM-CSF) and low-dose cytosine arabinoside. Each course of GM-CSF induced a rapid and tremendous increase in leukocyte count as well as in spleen size, 111-indium chloride scanning suggested a myeloid metaplasia of the spleen. This observation suggests that in some patients the granulopoietic response to the myeloid growth factor stimulation may be predominant in the spleen

  17. Methylation of histones in myeloid leukemias as a potential marker of granulocyte abnormalities

    Czech Academy of Sciences Publication Activity Database

    Lukášová, Emilie; Kořistek, Z.; Falk, Martin; Kozubek, Stanislav; Grigoryev, S.; Kozubek, Michal; Ondřej, Vladan; Kroupová, I.

    2005-01-01

    Roč. 77, č. 1 (2005), s. 1-12 ISSN 0741-5400 R&D Projects: GA MZd NC6987; GA AV ČR(CZ) IAA1065203; GA AV ČR(CZ) KSK5052113; GA AV ČR(CZ) IBS5004010; GA ČR(CZ) GA202/02/0804; GA MŠk ME 565 Institutional research plan: CEZ:AV0Z50040507 Keywords : human granulocytes differentiation * chromatin condensation * heterochromatin Subject RIV: BO - Biophysics Impact factor: 4.627, year: 2005

  18. STAT3 activation and infiltration of eosinophil granulocytes in mycosis fungoides

    DEFF Research Database (Denmark)

    Fredholm, Simon; Gjerdrum, Lise Mette R; Willerslev-Olsen, Andreas

    2014-01-01

    Eosinophil granulocytes have been implicated in anticancer immunity but recent data indicate that eosinophils can also promote cancer. Herein, we studied eosinophils in skin lesions from 43 patients with mycosis fungoides (MF). The presence of eosinophils correlated with disease stage: 78......% of patients with advanced disease displayed eosinophil infiltration, whereas this was only seen in 11% of patients with patches (p...) in malignant T-cells also stained positively for eosinophils, whereas this was only observed in 28% of pY-STAT3-negative patients (peosinophilic activation and trafficking factors: High-mobility group BOX-1 protein (HMGB1) and interleukin 5 (IL5). STAT3 si...

  19. Second case of chronic granulocytic leukemia with karyotypic evolution at acute crisis, occurring in so-called Nishiyama district

    Energy Technology Data Exchange (ETDEWEB)

    Yao, E; Tomonaga, Yu; Nishino, K; Matsunaga, M; Sadamori, N [Nagasaki Univ. (Japan). School of Medicine

    1978-09-01

    The whole process of a second case of chronic granulocytic leukemia in Nishiyama district where a very small amount of radiation existed for a long time was reported together with data measured by a human counter and the results of chromosomal analysis. No significantly high K or /sup 137/Cs values were measured by a human counter immediately after the onset. Chromosomal division aberration and chromosomal aberration, which seemed to be induced by radiation, also were not observed. However, granulocytic leukemia was diagnosed after chromosomal analysis of peripheral blood revealed Ph/sup 1/ chromosomes, white cell count increased, juvenile cells appeared, and basophil cells increased. Clinical features of typical chronic granulocytic leukemia in the exposed were observed during the chronic stage (7 years). In the acute stage, abnormal clones were discovered in all 16 chromosomes analyzed. Much karyotypic evolution identical to that in persons directly exposed to the A-bomb was also observed.

  20. Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

    Science.gov (United States)

    Bellotti, Marta; Salis, Annalisa; Grozio, Alessia; Damonte, Gianluca; Vigliarolo, Tiziana; Galatini, Andrea; Zocchi, Elena; Benatti, Umberto; Millo, Enrico

    2015-01-01

    The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    Science.gov (United States)

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

    Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID

  2. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Antje eFröhling

    2015-09-01

    Full Text Available Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfil the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results.The aim of this study was to compare the inactivation effects of peracetic acid (PAA, ozonated water (O3 and cold atmospheric pressure plasma (CAPP on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s with 0.25 % PAA at 10 °C, and after treatment (10 s with 3.8 mg l-1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 min and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l-1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process

  3. A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies

    International Nuclear Information System (INIS)

    Marcusson-Staahl, Maritha; Cederbrant, Karin

    2003-01-01

    A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51 Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems. Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5 ml peripheral blood, 1.25x10 5 effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled

  4. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

    Science.gov (United States)

    Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A

    2014-07-01

    X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

  5. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

    DEFF Research Database (Denmark)

    Ziebe, Søren; Loft, Anne; Povlsen, Betina B

    2013-01-01

    To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR).......To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR)....

  6. Hepatozoon ellisgreineri n. sp. (Hepatozoidae): description of the first avian apicomplexan blood parasite inhabiting granulocytes.

    Science.gov (United States)

    Valkiūnas, Gediminas; Mobley, Kristin; Iezhova, Tatjana A

    2016-02-01

    Blood parasites of the genus Hepatozoon (Apicomplexa, Hepatozoidae) infect all groups of terrestrial vertebrates, and particularly high prevalence and species diversity have been reported in reptiles and mammals. A few morphologically similar species, in which gamonts inhabit mononuclear leukocytes and red blood cells, have been described in birds. Here, we report a new Hepatozoon species, which was found in wild-caught secretary birds Sagittarius serpentarius, from Tanzania. Hepatozoon ellisgreineri n. sp. can be readily distinguished from all described species of avian Hepatozoon because its gamonts develop only in granulocytes, predominantly in heterophils, a unique characteristic among bird parasites of this genus. Additionally, this is the first reported avian apicomplexan blood parasite, which inhabits and matures in granulocytes. We describe H. ellisgreineri based on morphological characteristics of blood stages and their host cells. This finding broadens knowledge about host cells of avian Hepatozoon spp. and other avian apicomplexan blood parasites, contributing to the better understanding of the diversity of haematozoa. This is the first report of hepatozoonosis in endangered African birds of the Sagittariidae.

  7. The role of granulocyte macrophage-colony stimulating factor in gastrointestinal immunity to salmonellosis.

    Science.gov (United States)

    Coon, C; Beagley, K W; Bao, S

    2009-08-01

    Human Salmonella infection, in particular, typhoid fever is a highly infectious disease that remains a major public health problem causing significant morbidity and mortality. The outcome of these infections depends on the host's immune response, particularly the actions of granulocytes and macrophages. Using a mouse model of human typhoid fever, with Salmonella typhimurium infection of wild type and granulocyte macrophage-colony stimulating factor (GM-CSF) knock out mice we show a delay in the onset of immune-mediated tissue damage in the spleens and livers of GM-CSF(-/-) mice. Furthermore, GM-CSF(-/-) mice have a prolonged sequestration of S. typhimurium in affected tissues despite an increased production of F4/80+ effector cells. Moreover in the absence of GM-CSF, a decrease in pro-inflammatory cytokine expression of tumor necrosis factor-alpha, interleukin-12 (IL-12) and IL-18 was found, which may alter the host's immune response to infection. GM-CSF appears to play an important role in the pathogenesis of Salmonellosis, and may contribute significantly to the development of protective gastrointestinal mucosal immune responses against oral pathogens.

  8. Recombinant granulocyte colony-stimulating factor administered enterally to neonates is not absorbed.

    Science.gov (United States)

    Calhoun, Darlene A; Maheshwari, Akhil; Christensen, Robert D

    2003-08-01

    Granulocyte colony-stimulating factor (G-CSF) is present in liquids swallowed by the fetus and neonate; specifically, amniotic fluid, colostrum, and human milk. The swallowed G-CSF has local effects on enteric cells, which express the G-CSF receptor. However, some portion of the G-CSF ingested by the fetus and neonate might be absorbed into the circulation and have systemic actions, such as stimulating neutrophil production. To assess this possibility we sought to determine if circulating G-CSF concentrations of neonates increase after enteral administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). This was a single-center, prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhG-CSF (100 microg/kg) and 1 dose of placebo. Plasma G-CSF concentrations were measured at 2 and 4 hours after administration of the test solution. No significant change in plasma G-CSF concentration was observed after the enteral administration of rhG-CSF. On this basis, we conclude that orally administered rhG-CSF is not absorbed in significant quantities, and we speculate that the G-CSF swallowed by the fetus and neonate has local but not systemic effects.

  9. Sweet’s Syndrome Successfully Treated with Granulocyte and Monocyte Adsorption Apheresis

    Directory of Open Access Journals (Sweden)

    Asami Fujii

    2017-05-01

    Full Text Available Sweet’s syndrome is a neutrophilic dermatosis characterized by an abrupt onset of painful erythematous lesions showing neutrophilic infiltrates in the dermis. Fever and an elevated neutrophil level are generally observed. Sweet’s syndrome may be idiopathic, malignancy-associated, or drug-induced (mainly involving granulocyte colony-stimulating factor (G-CSF administration. Although systemic corticosteroids are usually effective, the symptoms of Sweet’s syndrome recur in some refractory cases. Herein, we report a case of a 55-year-old Japanese woman with recurrent symptoms of fever (>39°C and painful erythematous lesions on her four extremities, trunk, and neck. Laboratory findings revealed leukocytosis and high levels of C-reactive protein (CRP and G-CSF. She was diagnosed with a recurrence of Sweet’s syndrome, and was exclusively treated with granulocyte and monocyte adsorption apheresis (GMA therapy once a week for 3 consecutive weeks. After the first session of GMA therapy, all symptoms including the erythematous lesions and fever were completely resolved, and serum G-CSF level was reduced. Leukocyte count, neutrophil count, serum amyloid A protein, and CRP levels were restored within normal ranges by 2 weeks. Thus, GMA therapy can successfully treat a patient with recurrent Sweet’s syndrome, potentially related to the restoration of elevated serum G-CSF levels.

  10. Prevention of myelosuppression by combined treatment with enterosorbent and granulocyte colony-stimulating factor.

    Science.gov (United States)

    Shevchuk, O O; Posokhova, К А; Todor, I N; Lukianova, N Yu; Nikolaev, V G; Chekhun, V F

    2015-06-01

    Hematotoxicity and its complication are the prominent limiting factors for rational treatment of malignancies. Granulocyte colony-stimulating factor (G-CSF) is used to increase granulocyte production. It has been shown previously that enterosorption causes prominent myeloprotective activity also. Still, no trial was performed to combine both of them. To study the influence of combination of enterosorption and pharmaceutical analogue of naturally occurring G-CSF (filgrastim) on bone marrow protection and the growth of grafted tumor in a case of injection of melphalan (Mel). Mel injections were used for promotion of bone marrow suppression in rats. Carbon granulated enterosorbent C2 (IEPOR) was used for providing of enteral sorption detoxifying therapy. Filgrastim was used to increase white blood cells (WBC) count. The simultaneous usage of enterosorption and filgrastim had maximum effectiveness for restoring of all types of blood cells. WBC count was higher by 138.3% compared with the Mel group. The increase of platelets count by 98.5% was also observed. In the group (Mel + C2 + filgrastim) the absolute neutrophils count was twofold higher, in comparison with rats of Mel group. Simultaneous administration of G-CSF-analogue and carbonic enterosorbent C2 is a perspective approach for bone marrow protection, when the cytostatic drug melphalan is used. Such combination demonstrates prominent positive impact on restoring of all types of blood cells and had no influence on the antitumor efficacy.

  11. (±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Aam, B.B.; Fonnum, F.

    2006-01-01

    (±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic

  12. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Heidi A. Neubauer

    2017-03-01

    Full Text Available Sphingosine kinase 2 (SK2 is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs. Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout (Sphk2-/- MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

  13. Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

    Science.gov (United States)

    Galay, Remil Linggatong; Matsuo, Tomohide; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Kusakisako, Kodai; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2018-04-01

    Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method

    Science.gov (United States)

    Tabassum, Shahina; Al-Mahtab, Mamun; Nessa, Afzalun; Jahan, Munira; Shamim Kabir, Chowdhury Mohammad; Kamal, Mohammad; Cesar Aguilar, Julio

    2015-01-01

    Background Hepatitis B virus (HBV) infection has many faces. Precore and core promoter mutants resemble inactive carrier status. The identification of hepatitis B core antigen (HBcAg) in hepatocytes may have variable clinical significance. The present study was undertaken to detect HBcAg in chronic hepatitis B (CHB) patients and to assess the efficacy of detection system by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP). Materials and methods The study was done in 70 chronic HBV-infected patients. Out of 70 patients, eight (11.4%) were hepatitis B e antigen (HBeAg) positive and 62 (88.57%) were HBeAg negative. Hepatitis B core antigen was detected by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP) methods in liver tissue. Results All HBeAg positive patients expressed HBcAg by both IIF and IIP methods. Out of 62 patients with HBeAg-negative CHB, HBcAg was detected by IIF in 55 (88.7%) patients and by IIP in 51 (82.26%) patients. A positive relation among viral load and HBcAg detection was also found. This was more evident in the case of HBeAg negative patients and showed a positive relation with HBV DNA levels. Conclusion Hepatitis B core antigen can be detected using the IIF from formalin fixed paraffin block preparation and also by IIP method. This seems to reflect the magnitudes of HBV replication in CHB. How to cite this article Raihan R, Tabassum S, Al-Mahtab M, Nessa A, Jahan M, Kabir CMS, Kamal M, Aguilar JC. Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method. Euroasian J Hepato-Gastroenterol 2015;5(1):7-10. PMID:29201677

  15. Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

    International Nuclear Information System (INIS)

    Decho, Alan W; Beckman, Erin M; Chandler, G Thomas; Kawaguchi, Tomohiro

    2008-01-01

    An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae

  16. Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

    DEFF Research Database (Denmark)

    Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.

    2007-01-01

    The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

  17. Quantum-dot-based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneity

    International Nuclear Information System (INIS)

    Chen Chuang; Li Yan; Peng Jun; Xu Hao; Tang Hongwu; Zhang Zhiling; Pang Daiwen; Xia Heshun; Wu Qiongshui; Zeng Libo; Zhu Xiaobo

    2010-01-01

    Breast cancer (BC) is a heterogeneous tumor, and better understanding of its heterogeneity is essential to improving treatment effect. Quantum dot (QD)-based immunofluorescent nanotechnology (QD-IHC) for molecular pathology has potential advantages in delineating tumor heterogeneity. This potential is explored in this paper by QD-IHC imaging of HER2 and ER. BC heterogeneity can be displayed more clearly and sensitively by QD-IHC than conventional IHC in BC tissue microarrays. Furthermore, the simultaneous imaging of ER and HER2 might help understand their interactions during the process of evolution of heterogeneous BC.

  18. Fever of unknown origin: prospective comparison of diagnostic value of 18F-FDG PET and 111In-granulocyte scintigraphy

    DEFF Research Database (Denmark)

    Kjaer, Andreas; Lebech, Anne-Mette; Eigtved, Annika

    2004-01-01

    The diagnostic work-up in patients with fever of unknown origin (FUO) is often challenging and frequently includes nuclear medicine procedures. Whereas a role for leucocyte or granulocyte scintigraphy in FUO is generally accepted, a possible role of fluorine-18 fluorodeoxyglucose (FDG) positron...... emission tomography (PET) in these patients remains to be established. To study this, we compared prospectively, on a head-to-head basis, the diagnostic value of FDG-PET and indium-111 granulocyte scintigraphy in patients with FUO. Nineteen patients with FUO underwent both FDG-PET and (111)In......-granulocyte scintigraphy within 1 week. FDG-PET scans and granulocyte scintigrams were reviewed by different doctors who were blinded to the result of the other investigation. The diagnostic values of FDG-PET and granulocyte scintigraphy were evaluated with regard to identification of a focal infectious...

  19. Aloantígenos de granulocitos: Importancia clínica The granulocyte alloantigens. Clinical importance

    Directory of Open Access Journals (Sweden)

    María del Rosario López De Roux

    2003-12-01

    Full Text Available Los aloantígenos de granulocitos se agrupan en 2 grandes categorías: antígenos específicos de granulocitos y antígenos cuya distribución es más amplia y comprende otras líneas celulares. En 1998 se acordó establecer una nueva nomenclatura de los aloantígenos de granulocitos, basada en la localización glucoproteica de estos antígenos. La molécula FcgRIIIb es un miembro de la superfamilia de inmunoglobulinas (CD 16 en la cual se asientan varios de los aloantígenos específicos de granulocitos. Existen otros aloantígenos cuya función y localización se desconocen. Estas moléculas son de gran importancia clínica, pues se ven envueltas en una serie de enfermedades como la neutropenia neonatal aloinmune, cuyo carácter clínico moderado hace que pase inadvertida, la reacción febril no hemolítica, el daño pulmonar agudo relacionado con la transfusión, la neutropenia inmune asociada con el trasplante de médula ósea y la neutropenia autoinmune. Aunque se han producido avances en la caracterización de los aloantígenos de granulocitos, muchos puntos quedan sin aclarar, entre ellos, la significación clínica de muchos antígenos. El desarrollo creciente de técnicas moleculares, bioquímicas y serológicas para el estudio de los antígenos de células sanguíneas, nos permitirá aclarar los puntos que aún permanecen oscuros en este campo de la investigaciónThe granulocyte alloantigens are grouped into 2 big categories: specific granulocyte antigens and antigens, whose distribution is wider and comprises other cellular lines. In 1998, it was agreed to establish a new nomenclature of granulocyte alloantigens based on the glycoprotein localization of these antigens. The FcgRIIIb molecule is a member of the superfamily of immunoglobulins (CD 16, in which many of the specific granulocyte alloantigens are found. There are other alloantigens with an unknown function and localization. These molecules have a great clinical importance

  20. Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

    Science.gov (United States)

    Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

    2017-11-27

    Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

  1. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    Science.gov (United States)

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical Society.

  2. Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye.

    Science.gov (United States)

    Walpita, P; Darougar, S

    1989-07-01

    The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

  3. The effect of long-term treatment with granulocyte colony-stimulating factor on hematopoiesis in HIV-infected individuals

    DEFF Research Database (Denmark)

    Nielsen, S D; Sørensen, T U; Aladdin, H

    2000-01-01

    This randomized, placebo-controlled trial examine the long-term effect of granulocyte colony-stimulating factor (G-CSF) on absolute numbers of CD34+ progenitor cells and progenitor cell function in human immunodeficiency virus (HIV)-infected patients. G-CSF (300 microg filgrastim) or placebo was ...

  4. Aggressive cutaneous vasculitis in a patient with chronic lymphatic leukemia following granulocyte colony stimulating factor injection: a case report

    Directory of Open Access Journals (Sweden)

    El Husseiny Noha M

    2011-03-01

    Full Text Available Abstract Introduction Vasculitis has been reported in a few cases of chronic lymphatic leukemia and with granulocytic colony-stimulating factor therapy. Those with granulocytic colony-stimulating factor occurred after prolonged therapy and there was a rise in total leukocyte count unlike that in our patient who received just a single injection for the first time. Case presentation We report the case of a 64-year-old Egyptian man with chronic lymphatic leukemia who developed progressive cutaneous vasculitic lesions following injection of a single dose of a granulocytic colony stimulating factor before a third cycle of chemotherapy to improve neutropenia. This is an unusual case and the pathogenesis is not fully understood. Our patient was not on any medical treatment except for bisoprolol for ischemic heart disease. Although aggressive management with steroids, anticoagulation and plasmapheresis had been carried out, the condition was aggressive and the patient's consciousness deteriorated. A magnetic resonance imaging scan of his brain revealed multiple ischemic foci that could be attributed to vasculitis of the brain. Conclusion The aim of this case report is to highlight the importance of monitoring patients on granulocytic colony-stimulating factor therapy, especially in the context of other conditions (such as a hematological malignancy that may lead to an adverse outcome.

  5. CHOP compared with CHOP plus granulocyte colony-stimulating factor in elderly patients with aggressive non-Hodgkin's lymphoma

    NARCIS (Netherlands)

    Doorduijn, JK; van der Holt, B; van Imhoff, GW; van der Hem, KG; Kramer, MHH; van Oers, MHJ; Ossenkoppele, GJ; Verdonck, LF; Verhoef, GEG; Steijaert, MMC; Buijt, I.; Uyl-de Groot, CA; van Agthoven, M; Mulder, AH; Sonneveld, P; Schaafsma, M.

    2003-01-01

    Purpose : To investigate whether the relative close-intensity of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy could be improved by prophylactic administration of granulocyte colony-stimulating factor (G-CSF) in elderly patients with aggressive non-Hodgkin's lymphoma

  6. CHOP compared with CHOP plus granulocyte colony-stimulating factor in elderly patients with aggressive non-Hodgkin's lymphoma

    NARCIS (Netherlands)

    Doorduijn, J. K.; van der Holt, B.; van Imhoff, G. W.; van der Hem, K. G.; Kramer, M. H. H.; van Oers, M. H. J.; Ossenkoppele, G. J.; Schaafsma, M. R.; Verdonck, L. F.; Verhoef, G. E. G.; Steijaert, M. M. C.; Buijt, I.; Uyl-de Groot, C. A.; van Agthoven, M.; Mulder, A. H.; Sonneveld, P.

    2003-01-01

    PURPOSE: To investigate whether the relative dose-intensity of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy could be improved by prophylactic administration of granulocyte colony-stimulating factor (G-CSF) in elderly patients with aggressive non-Hodgkin's lymphoma

  7. Recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) treatment of clozapine-induced agranulocytosis

    DEFF Research Database (Denmark)

    Nielsen, H

    1993-01-01

    After 10 weeks of treatment with clozapine, severe agranulocytosis was diagnosed in a 33-year-old female. The patient was treated with filgrastim (granulocyte colony-stimulating factor [G-CSF]) 5 micrograms kg-1 day-1. The neutrophil count was 0.234 x 10(9) l-1 on admission, with a further decrease...

  8. Long-chain PUFA in Granulocytes, Mononuclear Cells, and RBC in Patients With Cystic Fibrosis: Relation to Liver Disease

    DEFF Research Database (Denmark)

    Jorgensen, Marianne H.; Ott, Peter; Michaelsen, Kim F.

    2012-01-01

    -related liver disease were matched with 20 CF patients without. Blood samples were analysed for liver biochemistry and haematology. Granulocytes, mononuclear cells, and RBC were separated by density gradient centrifugation, and fatty acid composition was measured by gas chromatography. Hepatic ultrasound...

  9. Stem cell mobilization by granulocyte colony-stimulating factor for myocardial recovery after acute myocardial infarction: a meta-analysis

    DEFF Research Database (Denmark)

    Zohlnhofer, D.; Dibra, A.; Koppara, T.

    2008-01-01

    OBJECTIVES: The objective of this meta-analysis was to evaluate the effect of stem cell mobilization by granulocyte colony-stimulating factor (G-CSF) on myocardial regeneration on the basis of a synthesis of the data generated by randomized, controlled clinical trials of G-CSF after acute...

  10. Increased production of granulocyte-macrophage colony-stimulating factor in Crohn's disease--a possible target for infliximab treatment

    DEFF Research Database (Denmark)

    Agnholt, Jørgen; Kelsen, Jens; Brandsborg, Birgitte

    2004-01-01

    The presence of neutrophils among epithelial cells is one of the major features of the inflammation in Crohn's disease, and has been used to indicate disease activity. The survival of neutrophils outside the blood vessels is limited and their longevity is influenced by granulocyte-macrophage colo...

  11. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A. [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil); Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E. [Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP (Brazil); DeSouza, I.A., E-mail: ivanidesouza@uol.com.br [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil)

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  12. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    International Nuclear Information System (INIS)

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A.; Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E.; DeSouza, I.A.

    2015-01-01

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  13. Aliphatic alcohols of illegally produced spirits can act synergistically on superoxide-anion production by human granulocytes.

    Science.gov (United States)

    Arnyas, Ervin M; Pál, László; Kovács, Csilla; Adány, Róza; McKee, Martin; Szűcs, Sándor

    2012-10-01

    Aliphatic alcohols present in illegally produced spirits in a large number of low and middle income countries have been implicated in the etiology of chronic liver disease and cirrhosis. Previous studies have confirmed that chronic alcoholism can lead to increased susceptibility to infectious diseases. Reduced superoxide-anion (O(2)·(-)) production by granulocytes could provide a mechanism by which antimicrobial defense is impaired in alcoholics. In vitro experiments have also demonstrated that ethanol can inhibit granulocyte O(2)·(-) generation. Aliphatic alcohols consumed as contaminants of illicit spirits may also influence O(2)·(-) production thereby contributing to a decrease in microbicidal activity. The aim of this study was to investigate this possibility. It measured the O(2)·(-) production by human granulocytes following treatment of the cells with aliphatic alcohol contaminants found in illicit spirits. Granulocytes were isolated from human buffy coats with centrifugal elutriation and then treated with individual aliphatic alcohols and their mixture. The O(2)·(-) production was stimulated with phorbol-12-13-dibutyrate and N-formyl-methionyl-leucyl-phenylalanine (FMLP) and measured by superoxide dismutase inhibitable reduction of ferricytochrome c. Aliphatic alcohols of illegally produced spirits inhibited the FMLP-induced O(2)·(-) production in a concentration dependent manner. They suppressed O(2)·(-) generation at 2.5-40 times lower concentrations when combined than when tested individually. Aliphatic alcohols found in illegally produced spirits can inhibit FMLP-induced O(2)·(-) production by granulocytes in a concentration-dependent manner. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit O(2)·(-) formation in heavy episodic drinkers.

  14. Granulocytic sarcoma masquerading as Ewing′s sarcoma: A diagnostic dilemma

    Directory of Open Access Journals (Sweden)

    Haresh Kunhi

    2008-01-01

    Full Text Available An eleven-year-old boy presented with a swelling in his left elbow. Radiologically the features were that of an Ewing′s sarcoma involving the ulna. Histopathology showed small round cell tumor strongly positive for Monoclonal Imperial Cancer research fund 2 (MIC2 antigen. Similar cells in the bone marrow were involved with MIC2 positivity. The patient developed skin lesions, which on biopsy were found to be chloromas. The initial biopsies were reevaluated with special stains revealing granulocytic sarcomas in acute myeloid leukemia masquerading as Ewing′s due to its MIC2 positivity. The possibility of myeloid neoplasms should be considered routinely with known MIC2 positive round cell tumors.

  15. Acute antibody-mediated rejection of skin grafts without involvement of granulocytes or complement

    International Nuclear Information System (INIS)

    Bogman, M.J.; Cornelissen, I.M.; Koene, R.A.

    1984-01-01

    In immunosuppressed mice that carry rat skin xeno-grafts, acute antibody-mediated graft rejection (AAR) can be induced by intravenous administration of mouse anti-rat globulin. Dependent on the amount of antibody injected and on the complement status of the recipient, an Arthus-like or a Shwartzman-like pattern of vasculitis occurs. The role of polymorphonuclear granulocytes (PMNs) in either type of vasculitis was tested by inducing AAR in recipients depleted of PMNs by total body irradiation. Despite the absence of PMNs in the graft vessels, AAR occurred both in the Arthus-like and in the Shwartzman-like type. Moreover, AAR could be elicited in PMN-depleted recipients that were complement-depleted by cobra venom factor treatment or were congenitally C5-deficient. We conclude that neither the PMN nor complement is an essential mediator the PMN nor complement is an essential mediator in this form of antibody-mediated vasculitis

  16. Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

    Science.gov (United States)

    Miyafusa, Takamitsu; Shibuya, Risa; Honda, Shinya

    2018-06-02

    Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 10 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Bone and bone-marrow blood flow in chronic granulocytic leukemia and primary myelofibrosis

    International Nuclear Information System (INIS)

    Lahtinen, R.; Lahtinen, T.; Romppanen, T.

    1982-01-01

    Blood flow in hematopoietic bone marrow and in nonhematopoietic bone has been measured with a Xe-133 washout method in 20 patients with chronic granulocytic leukemia (CGL) and in seven with primary myelofibrosis. Age-matched healthy persons served as controls. Bone-marrow blood flow in CGL was dependent upon the phase of the disease. In the metamorphosis phase, bone-marrow blood flow was high compared with that in the well-controlled phase. Apart from the initial phase, the mean values for bone blood flow in CGL were increased compared with the values of the healthy controls. In myelofibrosis the bone blood flow was also increased. Bone-marrow blood flow in these diseases was dependent upon the cellularity of bone marrow as measured morphometrically

  18. Aleukemic granulocytic sarcoma presenting at multiple sites: ovary, breast and soft tissue

    Directory of Open Access Journals (Sweden)

    Jitendra Singh Nigam

    2012-06-01

    Full Text Available An 18 year old female presented with the history of pain in abdomen, breast engorgement, swelling over both legs and breathlessness for three month. On clinical examination diagnosis of fibroadenoma breast was made. Ultrasonography of abdomen showed bilateral ovarian mass. Bilateral salpingo-ophrectomy was done and specimen was sent for histological examination. Two lobulated solid masses of tissues the larger one measuring 13x8x5 cm and smaller one measuring 10x7x5 cm in size received. Microscopic examination showed monomorphic population of discohesive, hyperchromatic small round cells had high N:C ratio, coarse chromatin, conspicuous nucleoli and scant to moderate amount of basophilic cytoplasm, lying in sheets and separated by fibrous strands and diffusely infiltrating the ovarian stroma. Fine needle aspiration from breast lump and leg swelling showed predominant population of blast cells. Myeloperoxidase was strongly positive and diagnosis of granulocytic sarcoma was confirmed.

  19. Hematologic improvement in dogs with parvovirus infection treated with recombinant canine granulocyte-colony stimulating factor.

    Science.gov (United States)

    Duffy, A; Dow, S; Ogilvie, G; Rao, S; Hackett, T

    2010-08-01

    Previously, dogs with canine parvovirus-induced neutropenia have not responded to treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF). However, recombinant canine G-CSF (rcG-CSF) has not been previously evaluated for treatment of parvovirus-induced neutropenia in dogs. We assessed the effectiveness of rcG-CSF in dogs with parvovirus-induced neutropenia with a prospective, open-label, nonrandomized clinical trial. Endpoints of our study were time to recovery of WBC and neutrophil counts, and duration of hospitalization. 28 dogs with parvovirus and neutropenia were treated with rcG-CSF and outcomes were compared to those of 34 dogs with parvovirus and neutropenia not treated with rcG-CSF. We found that mean WBC and neutrophil counts were significantly higher (P parvovirus infection, but indicate the need for additional studies to evaluate overall safety of the treatment.

  20. Promotion of Tumor Invasion by Cooperation of Granulocytes and Macrophages Activated by Anti-tumor Antibodies

    Directory of Open Access Journals (Sweden)

    Emilio Barbera-Guillem

    1999-11-01

    Full Text Available We investigated the potential role of anti-tumor antibodies and tumor antigens in the formation of immune complexes which promote matrix degradation and angiogenesis. B-cell deficient or B-cell depleted mice showed a reduction in tumor invasion and metastasis. In vitro invasion assays and in vivo models of metastasis showed that anti-sTn antibodies and sTn tumor antigens form complexes which induce granulocytes and macrophages together to mediate tumor invasion and metastasis by processes including extracellular matrix degradation and angiogenesis. These results suggest the existence of a tumor promoting role of a B-cell immune response induced by shed tumor associated antigens of solid, nonlymphoid tumors.

  1. Radiation-induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, D; Johnson, G R; Wilson, J [Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia)

    1977-01-01

    The peak sedimentation velocity of C/sub 57/BL mouse bone marrow progenitors of granulocytes and macrophages (GM-colony-forming cells, GM-CFC's) increased from 4.3 mm/h to 7 to 8 mm/h by 2 days after 250 rad whole body irradiation and slowly returned to normal over the next 3 weeks. Preliminary irradiation and/or endotoxin injection did not prevent this radiation-induced change. Some change in sedimentation velocity was seen with as little as 100 rad irradiation. Neither buoyant density nor cell cycle changes could account for the sedimentation velocity data which therefore indicate a major volume increase in the GM-CFC's. This size enlargement affected all subpopulations of GM-CFC's which consequently maintained their size relationship with one another.

  2. Treatment of chronic granulocytic leukemia by chemotherapy, total body irradiation and allogeneic bone marrow transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Doney, K; Buckner, C D; Sale, G E; Ramberg, R; Boyd, C; Thomas, E D [Fred Hutchinson Cancer Research Institute; Washington Univ., Seattle (USA). School of Medicine)

    1978-01-01

    Fourteen patients with chronic granulocytic leukemia received bone marrow grafts from HLA identical siblings. Ten patients were in blast crisis prior to grafting, three were in an accelerated phase of their disease, and one was aplastic secondary to chemotherapy. Prior to transplant all patients were conditioned with chemotherapy including cyclophosphamide plus 1,000 rad of total body irradiation. Ten patients achieved engraftment while four died 1 to 26 days after marrow infusion without functioning grafts. Two patients reveived a second infusion of donor marrow because of delayed engraftment. Neither marrow cell dose nor presence of myelofibrosis correlated with succesful engraftment. Three out of ten engrafted patients developed graft-versus-host disease. Interstitial pneumonia occurred in seven patients. The immediate cause of death was bacterial septicemia in six patients. All evidence of leukemia disappeared in nine out of ten evaluable patients. The median survival was 43 days. One patient had a complete remission of 16 months duration.

  3. Granulocyte Colony-Stimulating Factor in the Treatment of Acute Radiation Syndrome: A Concise Review

    Directory of Open Access Journals (Sweden)

    Michal Hofer

    2014-04-01

    Full Text Available This article concisely summarizes data on the action of one of the principal and best known growth factors, the granulocyte colony-stimulating factor (G-CSF, in a mammalian organism exposed to radiation doses inducing acute radiation syndrome. Highlighted are the topics of its real or anticipated use in radiation accident victims, the timing of its administration, the possibilities of combining G-CSF with other drugs, the ability of other agents to stimulate endogenous G-CSF production, as well as of the capability of this growth factor to ameliorate not only the bone marrow radiation syndrome but also the gastrointestinal radiation syndrome. G-CSF is one of the pivotal drugs in the treatment of radiation accident victims and its employment in this indication can be expected to remain or even grow in the future.

  4. Treatment of chronic granulocytic leukemia by chemotherapy, total body irradiation and allogeneic bone marrow transplantation

    International Nuclear Information System (INIS)

    Doney, K.; Buckner, C.D.; Sale, G.E.; Ramberg, R.; Boyd, C.; Thomas, E.D.; Washington Univ., Seattle

    1978-01-01

    Fourteen patients with chronic granulocytic leukemia received bone marrow grafts from HLA identical siblings. Ten patients were in blast crisis prior to grafting, three were in an accelerated phase of their disease, and one was aplastic secondary to chemotherapy. Prior to transplant all patients were conditioned with chemotherapy including cyclophosphamide plus 1,000 rad of total body irradiation. Ten patients achieved engraftment while four died 1 to 26 days after marrow infusion without functioning grafts. Two patients reveived a second infusion of donor marrow because of delayed engraftment. Neither marrow cell dose nor presence of myelofibrosis correlated with succesful engraftment. Three out of ten engrafted patients developed graft-versus-host disease. Interstitial pneumonia occurred in seven patients. The immediate cause of death was bacterial septicemia in six patients. All evidence of leukemia disappeared in nine out of ten evaluable patients. The median survival was 43 days. One patient had a complete remission of 16 months duration. (Author)

  5. Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

    Science.gov (United States)

    Manger, Ronald; Woodle, Doug; Berger, Andrew; Dickey, Robert W; Jester, Edward; Yasumoto, Takeshi; Lewis, Richard; Hawryluk, Timothy; Hungerford, James

    2014-01-01

    Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

  6. Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

    Directory of Open Access Journals (Sweden)

    Adam Z. Blatt

    2017-11-01

    Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

  7. Flow cytometric evaluation of peripheral blood and bone marrow and fine-needle aspirate samples from multiple sites in dogs with multicentric lymphoma.

    Science.gov (United States)

    Joetzke, Alexa E; Eberle, Nina; Nolte, Ingo; Mischke, Reinhard; Simon, Daniela

    2012-06-01

    To determine whether the extent of disease in dogs with lymphoma can be assessed via flow cytometry and to evaluate the suitability of fine-needle aspirates from the liver and spleen of dogs for flow cytometric examination. 44 dogs with multicentric B-cell (n = 35) or T-cell lymphoma (9) and 5 healthy control dogs. Procedures-Peripheral blood and bone marrow samples and fine-needle aspirates of lymph node, liver, and spleen were examined via flow cytometry. Logarithmically transformed T-cell-to-B-cell percentage ratio (log[T:B]) values were calculated. Thresholds defined by use of log(T:B) values of samples from control dogs were used to determine extranodal lymphoma involvement in lymphoma-affected dogs; results were compared with cytologic findings. 12 of 245 (5%) samples (9 liver, 1 spleen, and 2 bone marrow) had insufficient cellularity for flow cytometric evaluation. Mean log(T:B) values of samples from dogs with B-cell lymphoma were significantly lower than those of samples from the same site in dogs with T-cell lymphoma and in control dogs. In dogs with T-cell lymphoma, the log(T:B) of lymph node, bone marrow, and spleen samples was significantly higher than in control dogs. Of 165 samples assessed for extranodal lymphoma involvement, 116 (70%) tested positive via flow cytometric analysis; results agreed with cytologic findings in 133 of 161 (83%) samples evaluated via both methods. Results suggested that flow cytometry may aid in detection of extranodal lymphoma involvement in dogs, but further research is needed. Most fine-needle aspirates of liver and spleen were suitable for flow cytometric evaluation.

  8. Flow cytometric osmotic fragility test and eosin-5'-maleimide dye-binding tests are better than conventional osmotic fragility tests for the diagnosis of hereditary spherocytosis.

    Science.gov (United States)

    Arora, R D; Dass, J; Maydeo, S; Arya, V; Radhakrishnan, N; Sachdeva, A; Kotwal, J; Bhargava, M

    2018-03-24

    Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia with heterogeneous clinico-laboratory manifestations. We evaluated the flow-cytometric tests: eosin-5'-maleimide (EMA) and flow-cytometric osmotic fragility test (FOFT) and the conventional osmotic fragility tests (OFT) for the diagnosis of hereditary spherocytosis (HS). One hundred two suspected HS patients underwent EMA, FOFT, incubated OFT (IOFT), and room temperature OFT (RT-OFT). In addition, 10 cases of immune hemolytic anemia (IHA) were included, and performance of the above 4 tests was evaluated. For EMA and FOFT, 5 normal controls were assessed together with the patients and cutoffs were calculated using receiver-operator-characteristics curve (ROC) analysis. The best cutoff for %EMA decrease was 12.5%, and for FOFT, %residual red cells (%RRC) was 25.6%. The sensitivity and specificity of RT-OFT was 62.06% and 86.3%, respectively, while that of IOFT was 79.31% and 87.67%, respectively. Both flow cytometric tests performed better. Sensitivity and specificity of EMA was 86.2% and 93.9% respectively, and that of FOFT was 96.6% and 98.63%, respectively. The combination of the FOFT with IOFT or EMA dye-binding test yields a sensitivity of 100%, but with EMA, it had a higher specificity. Hb/MCHC was a predictor of the severity of the disease while %EMA decrease and %RRC did not correlate with severity of the disease. Flow-cytometric osmotic fragility test is the best possible single test followed by EMA for diagnosis of HS. A combination of FOFT and EMA can correctly diagnose 100% patients. These tests are likely to replace conventional OFTs in future. © 2018 John Wiley & Sons Ltd.

  9. Highly Expressed Granulocyte Colony-Stimulating Factor (G-CSF) and Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) in Human Gastric Cancer Leads to Poor Survival.

    Science.gov (United States)

    Fan, Zhisong; Li, Yong; Zhao, Qun; Fan, Liqiao; Tan, Bibo; Zuo, Jing; Hua, Kelei; Ji, Qiang

    2018-03-23

    BACKGROUND Chemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear. MATERIAL AND METHODS Seventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined. RESULTS The levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation. CONCLUSIONS These data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.

  10. A comparison of indirect immunofluorescence and electron microscopy for the diagnosis of some haemorrhagic viruses in cell cultures.

    Science.gov (United States)

    El Mekki, A A; van der Groen, G

    1981-09-01

    Yellow fever, dengue (types 1, 2 and 4), Chikungunya, Rift Valley fever, Ebola, Marburg, and Lassa viruses were inoculated into susceptible cell cultures and daily investigated by indirect immunofluorescence (IFA) and electron microscopy (EM) with a view to achieve an early detection-identification of these agents. Compared to the other cell lines tested (Vero, BHK-21 and Aedes albopictus), CV-1 cells were found to be more sensitive. Viral antigens were detected by IFA from a few hours post inoculation (CHIK and RVF) to a maximum of 3 days (YF and EBO). For most of the viruses studied, the cytopathic effect (CPE) commenced 2-3 days after the detection of viral antigens. Virus particles were detected by EM only in the case of EBO, MBG and LAS, before any CPE was observed in cell cultures.

  11. Comparison of immunofluorescence investigations and a new anti-DNA-antibody radioimmunoassay for the diagnosis of connective tissue diseases

    International Nuclear Information System (INIS)

    Neumeier, D.; Vogt, W.; Knedel, M.

    1976-01-01

    The procedure of determining the anti-DNA antibody activity is simplified by high-molecular double strand DNA labelled with 125 I. Cases of suspected connective tissue disease should first be examined by immunofluorescence microscopy, since this method can detect a wider spectrum of diseases with similar symptoms. For a differential diagnosis of SLE, the anti-DNA antibody activity is then investigated by a radioimmunoassay. When assessing the antibody activity, the following criteria should be kept in mind: - Findings of less than 10 units/ml serum do not indicate pathological changes, - Higher antibody activities up to 35 units/ml serum may occur in SLE patients but are also possible in other ANA positive diseases with similar symptoms, - Activities over 35 units/ml serum are nearly always a sign of SLE. (orig./GSE) [de

  12. Comparison of immunofluorescence investigations and a new anti-DNA-antibody radioimmunoassay for the diagnosis of connective tissue diseases

    Energy Technology Data Exchange (ETDEWEB)

    Neumeier, D; Vogt, W; Knedel, M

    1976-01-01

    The procedure of determining the anti-DNA antibody activity is simplified by high-molecular double strand DNA labelled with /sup 125/I. Cases of suspected connective tissue disease should first be examined by immunofluorescence microscopy, since this method can detect a wider spectrum of diseases with similar symptoms. For a differential diagnosis of SLE, the anti-DNA antibody activity is then investigated by a radioimmunoassay. When assessing the antibody activity, the following criteria should be kept in mind: - Findings of less than 10 units/ml serum do not indicate pathological changes, - Higher antibody activities up to 35 units/ml serum may occur in SLE patients but are also possible in other ANA positive diseases with similar symptoms, - Activities over 35 units/ml serum are nearly always a sign of SLE.

  13. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

    Directory of Open Access Journals (Sweden)

    Ondigo Bartholomew N

    2012-12-01

    Full Text Available Abstract Background Multiplex cytometric bead assay (CBA have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

  14. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis.

    Science.gov (United States)

    Suzuki, Hiroshi; Toyooka, Tatsushi; Ibuki, Yuko

    2007-04-15

    Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".

  15. Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

    Science.gov (United States)

    Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

    2012-01-01

    The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

  16. Microscopic and flow cytometric study of micronuclei in iododeoxyuridine labelled cells irradiated with soft X-rays

    International Nuclear Information System (INIS)

    Ludwikow, G.; Staalnacke, C.G.; Johanson, K.J.; Sundell-Bergman, S.; Richter, S.; Swedish Univ. of Agricultural Sciences, Uppsala; Uppsala Univ.

    1990-01-01

    Iododeoxyuridine labelled (IUdR(+)) and unlabelled (IUdR(-)) CHO cells irradiated with 2 Gy of soft X-rays showed only minor differences in the kinetics of micronuclei formation during the first 20 hours postirradiation period. Between 20 to 40 hours, the IUdR(-) cells showed approximately a constant number of micronuclei while the number of micronuclei in IUdR(+) cells was still increasing. The frequency of micronuclei was higher in IUdR(+) cells compared to IUdR(-) cells at 24 hours after irradiation with various doses up to 4.0 Gy. Dose modifying factors were found to be 1.3 (microscopic evaluation) and 1.8 (flow cytometric evaluation). Flow cytometry with use of two parameters, fluorescence from propidium iodide and light scattering, seems to be a good tool to estimate the frequency of micronuclei in CHO cells in the dose range up to about 4 Gy. At higher doses perturbation of the cell cycle and the appearance of dying cells will influence the results. (orig.)

  17. Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature.

    Science.gov (United States)

    Naik, Sangeeta Mahableshwar; Anil, Arga Chandrashekar

    2017-08-01

    Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (-80°C LN2 , -80°C, -20°C, and 5°C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16-19%), no cell loss was observed when samples were stored at 5°C. Cell recovery was not influenced by storage temperature until 4months but later samples preserved at -80°C LN2 , -80°C, and -20°C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5°C, autofluorescence was retained better at -80°C LN2 and -80°C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (-80°C LN2 and -80°C) while a higher concentration (1%) of GA when stored at a higher temperature (5°C) is recommended. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Immuno-flow cytometric detection of the ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense: independence of physiological state

    Science.gov (United States)

    Vrieling, Engel G.; van de Poll, Willem H.; Vriezekolk, Gertie; Gieskes, Winfried W. C.

    1997-05-01

    The ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense were cultured under different environmental conditions to test possible variability in immunochemical labelling intensity of cell-surface antigens using species-specific monoclonal antibodies. Variation of antigen abundance (which is directly related to labelling intensity) at the cell surface, determined by immuno-flow cytometry of cells labelled with FITC, appeared to be small but significant compared to control cultures. In general, a minor decrease in FIX fluorescence was recorded during exponential growth, followed by an increase during stationary growth. FITC fluorescence was correlated with cell size, shape and structure. This suggests a constant number of antigens per unit of cell surface. In all cultures, immunochemically labelled cells were distinguished clearly from unlabelled cells; immuno-flow cytometric identification is apparently not affected by growth conditions. Only at the end of the stationary growth phase in batch cultures did the FITC fluorescence values drop, which suggests that unhealthy, dying or lysing cells may either alter the composition of the cell surface or just fail to express the antigen.

  19. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  20. Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

    International Nuclear Information System (INIS)

    Jensen, R.H.; Bigbee, W.L.; Langlois, R.G.; Grant, S.G.; Pleshanov, P.G.; Chirkov, A.A.; Pilinskaya, M.A.

    1990-01-01

    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accident at Chernobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ionizing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure. 17 refs., 5 figs

  1. A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing Pathological Hitch

    Directory of Open Access Journals (Sweden)

    Puneet GANDHI

    2018-01-01

    Full Text Available Objective: Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging. Material and Method: Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation. Results: We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues. Conclusion: This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets.

  2. Changes in adhesion molecule expression and oxidative burst activity of granulocytes and monocytes during open-heart surgery with cardiopulmonary bypass compared with abdominal surgery

    DEFF Research Database (Denmark)

    Toft, P; Nielsen, C H; Tønnesen, Else Kirstine

    1998-01-01

    Cardiac and major abdominal surgery are associated with granulocytosis in peripheral blood. The purpose of the present study was to describe the granulocyte and monocyte oxidative burst and the expression of adhesion molecules following cardiac surgery with cardiopulmonary bypass and abdominal...... during cardiopulmonary bypass was observed. The percentage of CD11a-positive granulocytes increased from 30% pre-operatively to 75% following cardiopulmonary bypass, while CD44-positive granulocytes increased from 5% to 13%. Despite the extent of the changes, these were not significant. The oxidative...... to an increased per-operative oxidative burst activity, and the induction of adhesion molecules on granulocytes associated with the cardiopulmonary bypass and surgery. In conclusion, open-heart surgery with cardiopulmonary bypass was associated with a rapid and pronounced activation of leukocytes which may play...

  3. [The effect of lithium carbonate on the leukocyte count following ionizing radiation. 4. The effect of lithium carbonate on the activation of granulocytes].

    Science.gov (United States)

    Wolf, G; Müller, G M; Kehrberg, G

    1989-01-01

    From numerous investigations it is known that lithium carbonate promotes granulocytopoiesis by stimulation of CSF (colony stimulating factor) in bone marrow. To prove if no immature, in their functions restricted cells are delivered from bone marrow, the activity of granulocytes was tested in vitro in patients with lithium therapy. It could be seen that granulocytes of peripheral blood show an increased in-vitro-activation after lithium influence in vivo.

  4. Timing of granulocyte-colony stimulating factor treatment after acute myocardial infarction and recovery of left ventricular function: results from the STEMMI trial

    DEFF Research Database (Denmark)

    Overgaard, Mikkel; Ripa, Rasmus Sejersten; Wang, Yongzhong

    2010-01-01

    Granulocyte-colony stimulating factor (G-CSF) therapy after ST-elevation myocardial infarction (STEMI) have not demonstrated impact on systolic recovery compared to placebo. However, recent studies suggest that timing of G-CSF therapy is crucial.......Granulocyte-colony stimulating factor (G-CSF) therapy after ST-elevation myocardial infarction (STEMI) have not demonstrated impact on systolic recovery compared to placebo. However, recent studies suggest that timing of G-CSF therapy is crucial....

  5. Myeloprotective Action of Combined Application of Ukrainian Recombinant Granulocyte Colony Stimulating Factor (r-GCSF and Enterosorbent С2 in Rats with Malignant Guerin Carcinoma

    Directory of Open Access Journals (Sweden)

    Todor, I.M.

    2015-03-01

    Full Text Available The aim of the study is to analyze myeloprotective effect of novel enterosorbents alone and in combination with two recombinant granulocyte colony stimulating factors: Neupogen (Switzerland and r- GCSF (Ukraine. It is proven that Ukrainian version of recombinant granulocyte colony stimulating factor r-GCSF does not concede officinal drug Neupogen (Switzerland by its experimental therapeutic action and combined use with enterosorbent C2 significantly increases myeloprotective effect of both GCSF versions.

  6. A Randomized Case-Controlled Study of Recombinant Human Granulocyte Colony Stimulating Factor for the Treatment of Sepsis in Preterm Neutropenic Infants

    OpenAIRE

    Aktaş, Doğukan; Demirel, Bilge; Gürsoy, Tuğba; Ovalı, Fahri

    2015-01-01

    To investigate the efficacy and safety of recombinant human granulocyte colony-stimulating factor, recombinant human granulocyte-macrophage colony-stimulating factor (rhG-CSF) to treat sepsis in neutropenic preterm infants. Methods: Fifty-six neutropenic preterm infants with suspected or culture-proven sepsis hospitalized in Zeynep Kamil Maternity and Children's Educational and Training Hospital, Kozyatağı/Istanbul, Turkey between January 2008 and January 2010 were enrolled. Patients were ...

  7. Granulocytic Sarcoma by AML M4eo (inv16 after Allogeneic Stem Cell Transplantation without Bone Marrow Involvement

    Directory of Open Access Journals (Sweden)

    Stephan Zaenker

    2011-01-01

    Full Text Available Granulocytic sarcoma (GS represents a rare type of extramedullar manifestation from the acute myeloid leukaemia (AML. We report the case of a patient with recurrences of AML M4eo leukaemia in the uterus and the small intestine at 3 and 5 years, respectively, after matched related peripheral blood stem cell transplantation (PBSCT. The patient underwent the withdrawal of immunosuppression, hysterectomy, and local irradiation at first relapse, as well as systemic chemotherapy and donor lymphocyte infusions at second recurrence, inducing a second and third complete remission, respectively. At year six after transplantation, the patient experienced disease progression by meningeosis leukaemia to which she succumbed despite intrathecal chemotherapy. Following allogeneic stem cell transplantation, awareness for atypical manifestations of granulocytic sarcoma appears prudent, the cellular immunotherapy should aim at immunological disease control.

  8. Comparision of indium-111 oxinate labelled autologous granulocytes with indium-111 oxinate and indium-111 chloride as abscess scanning agents

    International Nuclear Information System (INIS)

    Goedemans, W.T.; Hardemann, M.R.; Belfer, A.J.

    1980-01-01

    Bacterial abscesses were evoked in goats. Imaging of these abscesses was obtained by means of labelling autologous granulocytes with 111 In oxinate, reinjection of the cells into the animal, and scintigraphy by gamma camera one day later. Comparable imaging results, however, were obtained after intravenous of 111 In oxinate or of 111 In chloride. The gamma camera images were supported by tissue distribution studies. In the case of administration of 111 In oxinate to the goats, the radioactivity accumulated in the cell fraction of the blood to a significant extent. This did not occur in the case of plain 111 In chloride. It remained unexplained why such different accumulation in cells did not result in differences in the scintigraphic studies. Blood clearance studies supplied conclusive evidence that the granulocytes stayed in the circulation for several days following labelling with 111 In oxinate and reinjection of the cells into the animals. (orig.) [de

  9. Flow Cytometric Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

    Science.gov (United States)

    Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2018-01-01

    In prion diseases, an abnormal isoform of prion protein (PrP Sc ) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP Sc -positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP Sc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP Sc -specific staining with monoclonal antibody (MAb) 132. The combination of PrP Sc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP Sc from those that were PrP Sc negative. The flow cytometric analysis of PrP Sc revealed the appearance of PrP Sc -positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP Sc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP Sc revealed a continuous increase in the proportion of PrP Sc -positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP Sc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP Sc -positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP Sc -associated pathogenesis. IMPORTANCE Although formation of PrP Sc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP Sc -associated pathogenesis, such as the intracerebral spread of PrP Sc and clearance of PrP Sc from the brain. Despite the great need for detailed analyses

  10. Granulocyte macrophage-colony-stimulating factor mouthwashes heal oral ulcers during head and neck radiotherapy

    International Nuclear Information System (INIS)

    Rovirosa, Angeles; Ferre, Jorge; Biete, Albert

    1998-01-01

    Purpose: To evaluate the effectiveness of granulocyte macrophage-colony-stimulating factor GM-CSF mouthwashes in the epithelization of radiation-induced oral mucosal ulceration, control of pain, and weight loss. Methods and Materials: Twelve patients received curative radiotherapy for head and neck carcinoma. All had oropharyngeal and/or oral mucosa irradiation, with a median dose of 72 Gy (range 50-74), with conventional fractionation. A total of 300 μg of GM-CSF in 250 cc of water for 1 h of mouthwashing was prescribed. The procedure started once oral ulceration in the irradiation field was detected. Patients, examined twice a week, were evaluated for oral ulceration, pain, and weight loss. Blood tests were taken weekly during GM-CSF administration. A comparison was carried out with 12 retrospective case-matched controls. Results: In the GM-CSF group, mucosa ulcerations healed in 9 of 12 (75%) of the patients during the course of the radiotherapy. Fifty percent of the patients said they felt less pain during the GM-CSF treatment; 30% needed morphine. The mean and median weight loss as a percentage of baseline weight in addition to the actual weight were 4.2% and 3%, respectively (variation ranged between a gain of 1% and a loss of 13%). No GM-CSF-related side effects were found. In the case control group, in the 12 cases, oral ulcerations increased during radiotherapy and two patients needed intubation intake and hospital admission, as opposed to the GM-CSF group. The mean and median percentage of weight loss were 5.8% and 5%, respectively. Sixty percent of patients needed morphine, as opposed to 30% in the GM-CSF group. Conclusions: Granulocyte macrophage-colony-stimulating factor was effective in curing mucosal ulcerations during the course of radiotherapy. This is the first time we have seen a drug with this capacity. Although the GM-CSF seems to be effective in the control of pain, oral intake, and weight loss, we need further studies with a greater number

  11. Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

    Science.gov (United States)

    Powell, Tiffany M; Paul, Jonathan D; Hill, Jonathan M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; McCoy, J Philip; Read, Elizabeth J; Khuu, Hanh M; Leitman, Susan F; Finkel, Toren; Cannon, Richard O

    2005-02-01

    Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair

  12. Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

    Science.gov (United States)

    Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

    2017-09-01

    Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  13. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Science.gov (United States)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  14. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    International Nuclear Information System (INIS)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-01-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ∼ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ∼ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ∼ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ∼ 12 nm retained bright fluorescence over an extended duration of ∼ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ∼ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ∼ 8.2% in human peripheral blood cells (PBMCs) which are CD33 low . The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  15. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Vanparys, Caroline, E-mail: caroline.vanparys@ua.ac.be [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)

    2010-09-15

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

  16. Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

    Science.gov (United States)

    Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

    2017-06-01

    Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p platelet count and IPC (both p-values Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 platelet turnover and COX-1 expression (all p-values platelet turnover and COX-1 and COX-2 expressions (all p-values platelet turnover in ITP patients (all p-values 0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in

  17. Histopathologic and Flow-Cytometric Analysis of Neoplastic and Benign “background” Tissue in Breast Carcinoma Resections

    Directory of Open Access Journals (Sweden)

    Daniel W. Visscher

    1998-01-01

    Full Text Available Two-color, multiparametric synthesis phase fraction (SPF analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb and malignant tissue samples (if available, SPFt from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1 tumor differentiation, (2 the proportion of tumor comprised of duct carcinoma-in situ (DCIS, and (3 the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases, atrophic (AT, 33% of cases, proliferative fibrocystic (PFC, 26% of cases, and non-proliferative fibrocystic (NPFC, 7% of cases. SPFt was inversely correlated with extent of DCIS (DCIS =0 – 20% tumor volume – 12.7% mean SPFt, vs. DCIS >20% tumor volume – 6.4% mean SPFt, p = 0.001. SPFt also correlated with the histology of background benign breast tissue (NTDLU – 14.8% mean SPFt vs. AT – 6.9% mean SPFt vs. PFC – 12.7% mean SPFt, p = 0.05 but it did not correlate with patient age or SPFb (overall mean =0.73%. SPFb was correlated with patient age (>56 yr – 0.59% mean SPFb vs. < yr – 0.84% mean SPFb, p = 0.02, with background histology (NTDLU – 1.1% mean SPFb vs. AT – 0.43% mean SPFb vs. PFC – 0.70% mean SPFb, p < 0.02 and with the grade of the neoplasm (well/moderate – 0.58% mean vs. poorly differentiated – 0.85% mean, p = 0.04. Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01.

  18. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    International Nuclear Information System (INIS)

    Vanparys, Caroline; Depiereux, Sophie; Nadzialek, Stephanie; Robbens, Johan; Blust, Ronny; Kestemont, Patrick; De Coen, Wim

    2010-01-01

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC 50 value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R 2 = 0.98), the estrogen receptor (ER) binding (R 2 = 0.84) and the ER transcription activation assay (R 2 = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of

  19. Flow cytometric assessment of microbial abundance in the near-field area of seawater reverse osmosis concentrate discharge

    KAUST Repository

    Van Der Merwe, Riaan

    2014-06-01

    The discharge of concentrate and other process waters from seawater reverse osmosis (SWRO) plant operations into the marine environment may adversely affect water quality in the near-field area surrounding the outfall. The main concerns are the increase in salt concentration in receiving waters, which results in a density increase and potential water stratification near the outfall, and possible increases in turbidity, e.g., due to the discharge of filter backwash waters. Changes in ambient water quality may affect microbial abundance in the area, for example by hindering the photosynthesis process or disrupting biogenesis. It is widely accepted that marine biodiversity is lower in more extreme conditions, such as high salinity environments. As aquatic microbial communities respond very rapidly to changes in their environment, they can be used as indicators for monitoring ambient water quality. The objective of this study was to assess possible changes in microbial abundance as a result of concentrate discharge into the near-field area (<. 25. m) surrounding the outfall of the King Abdullah University of Science and Technology (KAUST) SWRO plant. Flow cytometric (FCM) analysis was conducted in order to rapidly determine microbial abundance on a single-cell level in 107 samples, taken by diving, from the discharge area, the intake area and two control sites. FCM analysis combined the measurement of distinct scatter of cells and particles, autofluorescence of cyanobacteria and algae, and fluorescence after staining of nucleic acids with SYBR® Green for a total bacterial count. The results indicate that changes in microbial abundance in the near-field area of the KAUST SWRO outfall are minor and appear to be the result of a dilution effect rather than a direct impact of the concentrate discharge. © 2014 Elsevier B.V.

  20. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Energy Technology Data Exchange (ETDEWEB)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor, E-mail: manzoork@aims.amrita.edu, E-mail: ullasmony@aims.amrita.edu [Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041 (India)

    2011-07-15

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with {approx} 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of {approx} 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in {approx} 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of {approx} 12 nm retained bright fluorescence over an extended duration of {approx} a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of {approx} 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of {approx} 8.2% in human peripheral blood cells (PBMCs) which are CD33{sup low}. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  1. A quantum dot-immunofluorescent labeling method to investigate the interactions between a crinivirus and its whitefly vector

    Directory of Open Access Journals (Sweden)

    James C. K. Ng

    2013-04-01

    Full Text Available Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs, into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605 for biotin-conjugated anti-LIYV IgG (B-αIgG. IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated ∼7 to 8 fold slower than that of Alexa

  2. GRANULOCYTE INFILTRATION AND EXPRESSION OF THE PRO-ANGIOGENIC BV8 PROTEIN IN EXPERIMENTAL EL4 AND LEWIS LUNG CARCINOMA TUMORS.

    Science.gov (United States)

    Jiang, Kan; Kwak, Hyeongil; Tosato, Giovanna

    2013-01-18

    Although Vascular Endothelial Growth Factor (VEGF)-targeted therapies have shown efficacy in the treatment of certain advanced cancers, benefits to patients have been modest, which is attributed to tumor resistance to VEGF neutralization. Recent efforts to identify new targets to inhibit tumor angiogenesis have identified Bv8 (prokineticin 2), a myeloid cell-derived protein that promotes endothelial cell growth and tumor angiogenesis, but many mechanistic aspects of the pro-tumorigenic function of Bv8 are unclear. Here we demonstrate that CD11b+, Ly6C+, Ly6G+ granulocytes are the predominant cell source of Bv8 expression in bone marrow, spleen and in tumor tissues. Using granulocyte-deficient Growth factor independence-1 (Gfi1)-null mutant mice and normal littermates, we found that EL4 lymphoma tumors grow significantly larger in the granulocyte and Bv8-deficient mutant mice in comparison to the normal mice that display abundant tumor-associated granulocytes and Bv8 expression. Conversely, Lewis lung carcinoma (LLC-1) tumors grew to a significantly greater size in the normal mice in comparison to the Gfi1-null mice, but normal granulocyte tumor infiltration was modest. Quantitative analysis of tissue vascularization showed that EL4 and LLC-1 tumors from normal and Gfi1-mutant mice are similarly vascularized. These results confirm the critical contribution of the tumor microenvironment in determining the rate of tumor progression independently of tumor angiogenesis, and reveal some of the complexities of granulocyte and Bv8 functions in modulating tumor growth.

  3. Selective Granulocyte and Monocyte Apheresis as a Non-Pharmacological Option for Patients with Inflammatory Bowel Disease

    Science.gov (United States)

    C. Leitner, Gerda; Worel, Nina; Vogelsang, Harald

    2012-01-01

    Ulcerative colitis and Crohn's disease are the two most prevalent inflammatory bowel diseases. In both cases, the medically refractory and steroid-dependent type presents a therapeutic challenge. To help resolve this problem, a mainly Japanese team developed a new therapeutic option. There are two systems, both of which are able to selectively remove the main mediators of the disease, namely the activated pro-inflammatory cytokine-producing granulocytes and monocytes/macrophages, from the patient's blood circulation (GMA = granulocyte monocyte apheresis). One of the two systems is the Adacolumn® (Immunoresearch Laboratories, Takasaki, Japan) consisting of the ADA-monitor and a single-use column, which contains approximately 35,000 cellulose acetate beads. The exact mode of action is not yet sufficiently understood, but however, a modulation of the immune system takes place. As a result, less pro-inflammatory cytokines are released. Furthermore, the production of anti-inflammatory interleukin-1 receptor antagonist is increased, and the apoptosis of granulocytes boosted. The decreased LECAM-1-expression on leukocytes impedes the leukotaxis to the inflamed tissue, and CD10-negative immature granulocytes appear in the peripheral blood. Another effect to be mentioned is the removal of the peripheral dendritic cells and the leachate of regulatory T cells (T-regs). The second system is the Cellsorba® FX Filter (Asahi Medical, Tokyo, Japan). The range of efficiency, the indication, and the procedure are very similar to the Adacolumn. Solely the additional removal of lymphocytes can possibly limit the implementation since lymphopenia can increase the risk of autoimmune disease. Both systems provide a low-risk therapy with few adverse reactions. ASFA recommendations for GMA in inflammatory bowel disease are 2B due to the fact that not enough randomized double-blind studies are available to proof the efficacy of this treatment. PMID:22969694

  4. The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain

    OpenAIRE

    Giannini, Elisa; Lattanzi, Roberta; Nicotra, Annalisa; Campese, Antonio F.; Grazioli, Paola; Screpanti, Isabella; Balboni, Gianfranco; Salvadori, Severo; Sacerdote, Paola; Negri, Lucia

    2009-01-01

    Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the developme...

  5. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara

    2011-01-01

    Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associate...... significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema....

  6. Kinetic data of in-vivo labeled granulocytes in humans with a murine Tc-99m-labelled monoclonal antibody

    International Nuclear Information System (INIS)

    Becker, W.; Boerner, W.; Borst, U.; Schaefer, R.; Fischbach, W.; Pasurka, B.

    1989-01-01

    Twenty-five patients were examined in vivo with 99m Tc labelled monoclonal antibodies; 15 with suspected infections with an antigranulocyte antibody (BW 250/183), 10 with suspected recurrence of a colorectal carcinoma with an anti CEA antibody (BW 431/26). Both antibodies were IgG1 isotypes. In the patients with suspected infections no change of the peripheral leukocyte count could be observed after the antibody injection (1 mg, n=9; 0.05 mg, n=1; 0.25 mg, n=6). In 2 patients examined with the anti CEA antibody (2 mg), a significant decrease of the peripheral leukocyte count could be observed. The recovery rate of the 99m Tc antibody labelled granulocytes was calculated to be about 10%. The increase of the antibody-antigen binding was calculated to be 0.2%/min. In vivo the organ distribution curves demonstrated an increase of 99m Tc activity over spleen and bone marrow of 1.1%/min, which was interpreted as antigen-antibody reactivity. The organ distribution curves of the anti granulocyte antibody over spleen and bone marrow showed typical binding characteristics to the local granulocyte epitopes. The curves over other organs showed a simple perfusion pattern. The curves of the anti CEA antibody showed a perfusion pattern over all the examined organs. A sham dialysis model in one patient with renal insufficiency undergoing regular dialysis treatment demonstrated the viability of 99m Tc antibody labelled granulocytes in vivo. The kinetic patterns of the 99m Tc antibody in patients with Crohn's disease were interpreted as CEA binding of the antibody in the bowel wall. (orig.)

  7. Transplant of stem cells derived from bone marrow and granulocytic growth factor in acute and chronic ischemic myocardiopathy

    International Nuclear Information System (INIS)

    Senior Juan M; Cuellar Francisco; Velasquez Oscar; Velasquez Margarita; Navas Claudia M; Ortiz Sergio; Delgado Juan A; Guillerrno, Blanco; Londono Juan L; Coronado Manuel A; Gomez Francisco; Alzate, Fernando Leon; Zuluaga Alejandra

    2007-01-01

    Recent studies have shown the safety and efficacy of the stem cells derived from bone marrow (BMC) implant with concomitant administration of stimulating factor of granulocyte colonies in patients with acute myocardial infarction with ST segment elevation and in chronic ischemic cardiopathy. An open prospective (before and after) design was made to evaluate the safety and efficacy of cell therapy associated to growth factor administration. The first experience with this kind of therapy is reported. Methodology: this is a 6 months follow-up report of patients with acute and chronic ischemic cardiopathy to who transplant of stem cells derived from bone marrow mobilized with granulocyte colonies growth stimulating factor via coronary arteries or epicardium was realized. Two groups of patients were included: Ten patients with anterior wall infarct and 2. Five patients with chronic ischemic cardiopathy, all with extensive necrosis demonstrated by absence of myocardial viability through nuclear medicine and ejection fraction of less than 40%. Results: significant improvement of ejection fraction from 29.44 ± 3.36 to 37.6 ± 5.3 with p<0.001 and decrease of ventricular systolic and diastolic volume without statistical significance (p =0.31 and 0.4 respectively) were demonstrated. Exercise capacity evidenced by increment in the six minutes test, exercise time and the MET number achieved, increased in a significant way. There were significant changes in the perfusion defect from the second follow-up month and no complications directly related to the stem cells derived from bone marrow transplant or the use of stimulating granulocyte colony factor were presented. Conclusions: this is the first experience of stem cells derived from bone marrow transplant associated to the administration of stimulating granulocyte growth colony factor in which recovery of left ventricular function was demonstrated, as well as improvement in exercise capacity and in the perfusion defect

  8. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

    2003-01-01

    Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  9. Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sunami

    Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

  10. Granulocyte-Colony Stimulating Factor (G-CSF) for stroke: an individual patient data meta-analysis

    OpenAIRE

    England, Timothy J.; Sprigg, Nikola; Alasheev, Andrey M.; Belkin, Andrey A.; Kumar, Amit; Prasad, Kameshwar; Bath, Philip M.

    2016-01-01

    Granulocyte colony stimulating factor (G-CSF) may enhance recovery from stroke through neuroprotective mechanisms if administered early, or neurorepair if given later. Several small trials suggest administration is safe but effects on efficacy are unclear. We searched for randomised controlled trials (RCT) assessing G-CSF in patients with hyperacute, acute, subacute or chronic stroke, and asked Investigators to share individual patient data on baseline characteristics, stroke severity and typ...

  11. Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine synergize to enhance haematopoietic reconstitution in irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Pospisil, M.; Hofer, M.; Netikova, J.; Hola, J.; Vacek, A. [Academy of Sciences of the Czech Republic, Inst. of Biophysics, Brno (Czech Republic); Znojil, V.; Vacha, J. [Masaryk Univ., Medical Faculty, Brno (Czech Republic)

    1998-03-01

    The activation of adenosine receptors has recently been demonstrated to stimulate haematopoiesis. In the present study, we investigated the ability of drugs elevating extracellular adenosine to influence curative effects of granulocyte colony-stimulating factor (G-CSF) in mice exposed to a sublethal dose of 4 Gy of {sup 60}Co radiation. Elevation of extracellular adenosine in mice was induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole plus AMP, and G-CSF, administered either alone or in combination, were evaluated. The drugs were injected to mice in a 4-d treatment regimen starting on d 3 after irradiation and the haematopoietic response was evaluated on d 7, 10, 14, 18 and 24 after irradiation. While the effects of G-CSF on the late maturation stages of blood cells, appearing shortly after the completion of the treatment, were not influenced by dipyridamole plus AMP, positive effects of the combination therapy occurred in the post-irradiation recovery phase which is dependent on the repopulation of haematopoietic stem cells. This was indicated by the significant elevation of counts of granulocyte-macrophage progenitor cells (GM-CFC) and granulocytic cells in the bone marrow (d 14), of GM-CFC (d 14), granulocytic and erythroid cells (d 14 and 18) in the spleen, and of neutrophils (d 18), monocytes (d 14 and 18) and platelets (d 18) in the peripheral blood. These effects suggest that the repopulation potential of the combination therapy lies in a common multi-lineage cell population. The results of this study implicate the promising possibility to enhance the curative effects of G-CSF under conditions of myelosuppressive state induced by radiation exposure. (au) 43 refs.

  12. Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine synergize to enhance haematopoietic reconstitution in irradiated mice

    International Nuclear Information System (INIS)

    Pospisil, M.; Hofer, M.; Netikova, J.; Hola, J.; Vacek, A.; Znojil, V.; Vacha, J.

    1998-01-01

    The activation of adenosine receptors has recently been demonstrated to stimulate haematopoiesis. In the present study, we investigated the ability of drugs elevating extracellular adenosine to influence curative effects of granulocyte colony-stimulating factor (G-CSF) in mice exposed to a sublethal dose of 4 Gy of 60 Co radiation. Elevation of extracellular adenosine in mice was induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole plus AMP, and G-CSF, administered either alone or in combination, were evaluated. The drugs were injected to mice in a 4-d treatment regimen starting on d 3 after irradiation and the haematopoietic response was evaluated on d 7, 10, 14, 18 and 24 after irradiation. While the effects of G-CSF on the late maturation stages of blood cells, appearing shortly after the completion of the treatment, were not influenced by dipyridamole plus AMP, positive effects of the combination therapy occurred in the post-irradiation recovery phase which is dependent on the repopulation of haematopoietic stem cells. This was indicated by the significant elevation of counts of granulocyte-macrophage progenitor cells (GM-CFC) and granulocytic cells in the bone marrow (d 14), of GM-CFC (d 14), granulocytic and erythroid cells (d 14 and 18) in the spleen, and of neutrophils (d 18), monocytes (d 14 and 18) and platelets (d 18) in the peripheral blood. These effects suggest that the repopulation potential of the combination therapy lies in a common multi-lineage cell population. The results of this study implicate the promising possibility to enhance the curative effects of G-CSF under conditions of myelosuppressive state induced by radiation exposure. (au)

  13. Granulocyte-Monocyte Apheresis in Steroid-Dependent, Azathioprine-Intolerant/Resistant Moderate Ulcerative Colitis: A Prospective Multicenter Study

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    Gianni Imperiali

    2017-01-01

    Full Text Available Background. Granulocyte-monocyte apheresis has been proposed for the treatment of ulcerative colitis, although it is limited by costs and variability of results. Aim. To assess effectiveness of granulocyte-monocyte apheresis in patients with steroid-dependent, azathioprine-intolerant/resistant moderate ulcerative colitis. Methods. Consecutive patients fulfilling inclusion criteria were prospectively enrolled, treated by apheresis, and followed up for 12 months. The primary end point of the study was steroid-free clinical remission at 12 months, with no need for biologic therapy or surgery. Results. From January to December 2013, 33 patients were enrolled. After one year of follow-up, 12 (36% patients had clinical remission, were steroid-free, and had no need for biological therapy or surgery; 3 (9% cases showed a clinical response (but not clinical remission. Moreover, 12 (36% patients required biologic therapy, 4 (12% underwent colectomy, and in the other 2 (6% a reduction, but not withdrawal, of steroid dose was achieved. Conclusions. Our study shows that a standard course of granulocyte-monocyte apheresis is associated with a 36% steroid-free clinical remission in patients with steroid-dependent, azathioprine-intolerant or resistant moderate ulcerative colitis. Apheresis might represent an alternative to biologic therapy or surgery in this specific subgroup of patients. This trial is registered with Clinicaltrial.gov NCT03189888.

  14. CD64 on monocytes and granulocytes in severe acute bronchiolitis: Pilot study on its usefulness as a bacterial infection biomarker.

    Science.gov (United States)

    García-Salido, Alberto; Serrano-González, Ana; Casado-Flores, Juan; Sierra-Colomina, Montserrat; de Azagra-Garde, Amelia Martínez; García-Teresa, María Ángeles; Melen, Gustavo J; Ramírez-Orellana, Manuel

    2018-02-27

    The CD64 receptor has been described as a biomarker of bacterial infection. We speculated that CD64 surface expression on monocytes and granulocytes of children with severe acute bronchiolitis (SAB) could be altered in cases of probable bacterial infection (PBI) determined using classical biomarkers (procalcitonin and C-reactive protein, leukocyte count, and radiographic findings). A prospective observational pilot study was conducted from October 2015 to February 2016 in children admitted for pediatric critical care. A blood sample was taken in the first 24 hours of admission, and CD64 was measured by flow cytometry. The values obtained were analyzed and correlated with traditional biomarkers of PBI. Thirty-two children were included; a correlation was found between CD64 expression and the PBI criteria. CD64 surface expression was higher in children with PBI (area under the receiver operating characteristic curve of 0.73; P = 0.042) and the percentage of CD64 + granulocytes was higher in children with PBI. This is the first study to describe CD64 surface expression on monocytes and granulocytes in SAB, finding CD64 values to be higher in children with PBI. Larger clinical studies are needed to elucidate the real accuracy of CD64 as a biomarker of bacterial infection. ©2018 Society for Leukocyte Biology.

  15. New mononuclear leukocyte-like populations within the granulocyte scatter gate detected by flow cytometry (Conference Presentation)

    Science.gov (United States)

    Melzer, Susanne; Löffler, Markus; Kautzner, Marlene; Tárnok, Attila

    2017-02-01

    Granulocytes are the major players in innate immunity and are prognostic markers in diseases. An in-depth phenotypic characterization of granulocyte subtypes and correlation with biometry or lifestyle is so far lacking. The reason is, that either preparation of mononuclear cells was analyzed or that cells in the neutrophil window were neglected in the analysis. Here we show for the first time lymphocyte- (LL) and monocyte-like (ML) cells within the granulocyte scatter gate as new, previously unknown cell subpopulation. Immunophenotyping of 905 healthy German adults from the LIFE study [1] was performed by 10-color flow cytometry [2]. Age of men (n=420): 56.5±14.0 years, women (n=485): 56.7±13.6 y (range of 18-81 y). Data analyzed by FlowJo v10.0.6. Values compared by Mann-Whitney-U test: men vs women, young (18-49 y) vs. elderly (50-81 y.) men, and young (19-49 y.) vs. elderly (50-81 y.) women; significance: page, except LL2 in women. In conclusion, new lymphocyte like cell types with the neutrophil scatter characteristics are reported. Counts correlate with age and gender. We plan to sort these new subtypes for further functional characterization and aim to establish them as cellular biomarkers for the early detection of various diseases. [1] BMC Public Health. 2015;15:691; [2] Cytometry A. 2014;85(9):781

  16. An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix

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    Omar S. Qureshi

    2017-10-01

    Full Text Available Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

  17. Characterisation of L-Type Amino Acid Transporter 1 (LAT1 Expression in Human Skeletal Muscle by Immunofluorescent Microscopy

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    Nathan Hodson

    2017-12-01

    Full Text Available The branch chain amino acid leucine is a potent stimulator of protein synthesis in skeletal muscle. Leucine rapidly enters the cell via the L-Type Amino Acid Transporter 1 (LAT1; however, little is known regarding the localisation and distribution of this transporter in human skeletal muscle. Therefore, we applied immunofluorescence staining approaches to visualise LAT1 in wild type (WT and LAT1 muscle-specific knockout (mKO mice, in addition to basal human skeletal muscle samples. LAT1 positive staining was visually greater in WT muscles compared to mKO muscle. In human skeletal muscle, positive LAT1 staining was noted close to the sarcolemmal membrane (dystrophin positive staining, with a greater staining intensity for LAT1 observed in the sarcoplasmic regions of type II fibres (those not stained positively for myosin heavy-chain 1, Type II—25.07 ± 5.93, Type I—13.71 ± 1.98, p < 0.01, suggesting a greater abundance of this protein in these fibres. Finally, we observed association with LAT1 and endothelial nitric oxide synthase (eNOS, suggesting LAT1 association close to the microvasculature. This is the first study to visualise the distribution and localisation of LAT1 in human skeletal muscle. As such, this approach provides a validated experimental platform to study the role and regulation of LAT1 in human skeletal muscle in response to various physiological and pathophysiological models.

  18. Direct immunofluorescence assay compared to cell culture for the diagnosis of mucocutaneous herpes simplex virus infections in children.

    Science.gov (United States)

    Caviness, A Chantal; Oelze, Lindsay L; Saz, Ulas E; Greer, Jewel M; Demmler-Harrison, Gail J

    2010-09-01

    Direct immunofluorescence assay (DFA) is commonly used for the rapid identification of herpes simplex virus (HSV) infection in mucocutaneous lesions, yet little is known about its diagnostic accuracy. To determine the diagnostic yield and accuracy of HSV DFA for the diagnosis of mucocutaneous HSV infection in pediatric patients. Retrospective cross-sectional study of all patients who underwent HSV DFA testing by the Texas Children's Hospital Diagnostic Virology between January 1, 1995 and December 31, 2005. HSV DFA sensitivity, specificity, positive likelihood ratio (LRs), and negative LRs were estimated using viral culture as the reference standard. 659 specimens were submitted for HSV DFA with concurrent viral cultures. Viral cultures were positive for HSV type 1 in 158 (24%) and HSV type 2 in 2 (0.3%). There were 433 different patients with a median age of 8.6 years. Types of lesions were as follows: 50% ulcerative, 26% vesicular, 8% erythema or purpura, 5% pustular, and 11% missing. Of the 659 specimens submitted for HSV DFA, 160 (24%) were inconclusive due to inadequate cells. Of the 499 adequate specimens, overall HSV DFA test accuracy was: sensitivity 61%, specificity 99%, LR positive 40, and LR negative 0.39. A quarter of specimens submitted for HSV DFA testing are not adequate for DFA testing. When HSV DFA can be performed, it is specific, but not sensitive, for the identification of mucocutaneous HSV infection in children. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  19. DETECTION OF ANTIBODIES TO CANDIDA ALBICANS GERM TUBE BY IMMUNOFLUORESCENCE IN IMMUNOSUPPRESSED MICE WITH EXPERIMENTAL SYSTEMIC CANDIDIASIS

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    F. Zaini

    2007-07-01

    Full Text Available "nThe increasing incidence of systemic candidiasis, which parallels the use of invasive and immunosuppressive medical procedures, necessitates development of rapid and cost effective tests for diagnosis of systemic candidiasis. Therefore in this study 85 mice were first immunosuppressed by cyclophosphamide and then infected by Candida albicans NCPF 3153. Other 85 mice were employed as control. The case and control mice were bled and then autopsied. Hearts and kidneys were checked by direct, histopathological and cultural examination for systemic candidiasis. The 85 sera from histological proven cases and 85 control mice were adsorbed with heat killed blastospores of same strain of C. albicans. Anti-Candida albicans germ tube antibodies were detected by indirect immunofluorescence assay for diagnosis of invasive candidiasis in case and control mice. In addition, sera from 35 mice with proven cryptococcosis were also tested. While 84 mice with proven systemic candidiasis (100% had anti-germ tube antibodies, these antibodies were absent in all controls and mice with cryptococcosis. The specificity was 100%, indicating a high degree of discrimination was possible between systemic candidiasis and cryptococcosis in the mice studied. It must be concluded that anti-germ tube responses did not appear to be significantly reduced in immunocompromised mice.

  20. Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations.

    Science.gov (United States)

    Kumar, Ashok; Yerneni, Lakshmana K

    2009-01-01

    Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.

  1. Performance of an ELISA and Indirect Immunofluorescence Assay in Serological Diagnosis of Zoonotic Cutaneous Leishmaniasis in Iran

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    Bahador Sarkari

    2014-01-01

    Full Text Available Serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (VL and considered as a routine method for diagnosis of VL while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (CL. This study aimed to assess the performance of indirect immunofluorescent-antibody test (IFA and enzyme-linked immunosorbent assay (ELISA for serodiagnosis of cutaneous leishmaniasis in Iran. Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera from healthy controls along with 50 sera from non-CL patients were collected. Antigen was prepared from promastigotes and amastigotes of Leishmania major. IFA was used to detect anti-Leishmania IgG while ELISA was used to detect anti-Leishmania IgM, total IgG, or IgG subclasses (IgG1 and 4. ELISA, for detection of total IgG and IgM, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. Sensitivity and specificity of ELISA for detecting IgG1 and IgG4 were 64%, 75% and 85%, 49%, respectively. Sensitivity and specificity of IFA were 91.6% and 81%. Conclusion. Findings of this study demonstrated that serological test, especially IFA, can be used for proper diagnosis of CL.

  2. Diagnostic utility of a direct immunofluorescence test to detect feline coronavirus antigen in macrophages in effusive feline infectious peritonitis.

    Science.gov (United States)

    Litster, A L; Pogranichniy, R; Lin, T-L

    2013-11-01

    The antemortem diagnosis of feline infectious peritonitis (FIP) remains challenging in clinical practice, since current testing methods have suboptimal diagnostic accuracy. Immunohistochemical testing of biopsy specimens and postmortem examination are the standard diagnostic methods, although direct immunofluorescence (DIF) testing to detect feline coronavirus in macrophages in effusion specimens has been reported to have 100% specificity and has been recommended as an antemortem confirmatory test. The aim of this study was to compare the results of DIF testing in antemortem feline effusions with postmortem results using field samples. Effusion specimens were collected antemortem from 17 cats and tested by DIF, followed by postmortem examination. Histopathological examination of specimens collected at postmortem confirmed FIP in 10/17 cases and ruled out FIP out in 7/17 cases. Antemortem DIF testing was positive in all 10 cases confirmed as FIP at postmortem examination. In the seven cats where FIP was ruled out at postmortem examination, DIF was negative in five cases and positive in the remaining two cases. The calculated sensitivity of DIF testing was 100% and the specificity was 71.4%. Duplicate effusion specimens from eight cats that were initially DIF positive were stored refrigerated (4 °C) or at room temperature (22-25 °C) and subjected to serial DIF testing to determine the duration of positive results. DIF-positive specimens stored at both temperatures retained their positive status for at least 2 days. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Surface-biofunctionalized multicore/shell CdTe@SiO2 composite particles for immunofluorescence assay

    Science.gov (United States)

    Jing, Lihong; Li, Yilin; Ding, Ke; Qiao, Ruirui; Rogach, Andrey L.; Gao, Mingyuan

    2011-12-01

    Strongly fluorescent multicore/shell structured CdTe@SiO2 composite particles of ~ 50 nm were synthesized via the reverse microemulsion method by using CdTe quantum dots co-stabilized by thioglycolic acid and thioglycerol. The optical stability of the CdTe@SiO2 composite particles in a wide pH range, under prolonged UV irradiation in pure water, or in different types of physiological buffers was systematically investigated. Towards immunofluorescence assay, both poly(ethylene glycol) (PEG) and carboxyl residues were simultaneously grafted on the surface of the silanol-terminated CdTe@SiO2 composite particles upon further reactions with silane reagents bearing a PEG segment and carboxyl group, respectively, in order to suppress the nonspecific interactions of the silica particles with proteins and meanwhile introduce reactive moieties to the fluorescent particles. Agarose gel electrophoresis, dynamic light scattering and conventional optical spectroscopy were combined to investigate the effectiveness of the surface modifications. Via the surface carboxyl residue, various antibodies were covalently conjugated to the fluorescent particles and the resultant fluorescent probes were used in detecting cancer cells through both direct fluorescent antibody and indirect fluorescent antibody assays, respectively.

  4. Immunofluorescent histological studies of the role of fibronectin in the expression of the associative preferences of embryonic tissues.

    Science.gov (United States)

    Armstrong, P B; Armstrong, M T

    1981-08-01

    The identity of the chemical factors controlling the spreading behaviour of sheets of cells was examined in organ culture. When aggregates of two dissimilar tissues are apposed in organ culture, one tissue spreads reproducibly over the surface of the second. The present study employed indirect immunofluorescent localization techniques to evaluate the hypothesis that the spreading behaviour of chick embryonic heart tissue in culture is dominated by the presence or absence of the cell-surface and extracellular matrix protein fibronectin in the surface layers of the aggregates. Specifically, the hypothesis proposes that aggregates that display surface fibronectin earlier after culturing and/or in higher quantities segregate internally to aggregates that are slower to develop a surface layer of fibronectin or in which this layer contains reduced amounts of fibronectin. The hypothesis has been supported for 3 categories of behaviour of chick embryo heart tissue: (1) myocyte aggregates spread over myocyte aggregates containing a 20% admixture of heart fibroblasts, which in turn spread over heart fibroblast aggregates; (2) 5-day embryonic ventricle-tissue fragments maintained in culture for 0.5 days spread over ventricle fragments cultured for 2.5 days; and (3) 2-day embryonic ventricle spreads over 5-day ventricle. In all these situations, the aggregate type that segregates to an internal position displays more fibronectin at its surface than aggregate types that spread to occupy an external position. Evidence is presented that the fibronectin in heart tissue aggregates is elaborated by heart fibroblasts.

  5. In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis

    International Nuclear Information System (INIS)

    Aglietta, M.; Monzeglio, C.; Sanavio, F.; Apra, F.; Morelli, S.; Stacchini, A.; Piacibello, W.; Bussolino, F.; Bagnara, G.; Zauli, G.

    1991-01-01

    The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production

  6. A comparative study of total body irradiation as a method of inducing granulocyte depletion in mice

    International Nuclear Information System (INIS)

    Bogman, M.J.J.T.; Cornelissen, I.M.H.A.; Berden, J.H.M.; Jong, J. de; Koene, R.A.P.

    1984-01-01

    Since conventional methods of inducing depletion of polymorphonuclear granulocytes (PMNs) in mice, such as treatment with cytostatic drugs and anti-PMN sera, proved to be insufficient to induce a stable PMN depletion for several days, and were accompanied by considerable toxic side effects, we induced neutrophil depletion in mice by total body irradiation (TBI) in a single dose of 6.0 Gy (600 rads.) at a dose rate of 0.20 Gy/min. This treatment reduced the number of PMNs in the peripheral circulation to values below 150/μl from day 3-10 after irradiation. The number of lymphocytes fell simultaneously. Platelet counts remained above 60% of normal values during the first 7 days after irradiation. Complement levels were not significantly affected by TBI. The results show that TBI of 6.0 Gy induces pronounced and stable PMN depletion in mice for at least 7 days. Furthermore, under an aseptic regimen the mice can be kept in good condition and losses are less than 5%. (Auth.)

  7. Long-active granulocyte colony-stimulating factor for peripheral blood hematopoietic progenitor cell mobilization.

    Science.gov (United States)

    Martino, Massimo; Laszlo, Daniele; Lanza, Francesco

    2014-06-01

    Peg-filgrastim (PEG-FIL), a polyethylene glycol-conjugated form of granulocyte colony-stimulating factor (G-CSF), has been introduced in clinical practice and is effective in shortening the time of neutropenia after cytotoxic chemotherapy. G-CSF has emerged as the preferred cytokine for hematopoietic progenitor cells' (HPC) mobilization. Nevertheless, data on the ability of PEG-FIL in this field have been published. We review publications in the field with the goal of providing an overview of this approach. PEG-FIL may be able to mobilize CD34(+) cells in a more timely fashion than G-CSF, with the advantages of only a single-dose administration, an earlier start and a reduction in the number of apheresis procedures. The main controversies concern the dosage of the drug and the optimal dose. In the context of chemo-mobilization, a single dose of 6 mg PEG-FIL seems effective in terms of HPC's mobilization and there is no increase in this effect if the dose is doubled to 12 mg. Steady-state mobilization requires higher doses of PEG-FIL and this approach is not cost-effective when compared with G-CSF. The experiences with PEG-FIL in the healthy donor setting are very limited.

  8. Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

    Science.gov (United States)

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

  9. Granulocyte-colony stimulating factor controls neural and behavioral plasticity in response to cocaine.

    Science.gov (United States)

    Calipari, Erin S; Godino, Arthur; Peck, Emily G; Salery, Marine; Mervosh, Nicholas L; Landry, Joseph A; Russo, Scott J; Hurd, Yasmin L; Nestler, Eric J; Kiraly, Drew D

    2018-01-16

    Cocaine addiction is characterized by dysfunction in reward-related brain circuits, leading to maladaptive motivation to seek and take the drug. There are currently no clinically available pharmacotherapies to treat cocaine addiction. Through a broad screen of innate immune mediators, we identify granulocyte-colony stimulating factor (G-CSF) as a potent mediator of cocaine-induced adaptations. Here we report that G-CSF potentiates cocaine-induced increases in neural activity in the nucleus accumbens (NAc) and prefrontal cortex. In addition, G-CSF injections potentiate cocaine place preference and enhance motivation to self-administer cocaine, while not affecting responses to natural rewards. Infusion of G-CSF neutralizing antibody into NAc blocks the ability of G-CSF to modulate cocaine's behavioral effects, providing a direct link between central G-CSF action in NAc and cocaine reward. These results demonstrate that manipulating G-CSF is sufficient to alter the motivation for cocaine, but not natural rewards, providing a pharmacotherapeutic avenue to manipulate addictive behaviors without abuse potential.

  10. A scanning electron microscopic study of 34 cases of acute granulocytic, myelomonocytic, monoblastic and histiocytic leukemia.

    Science.gov (United States)

    Polliack, A; McKenzie, S; Gee, T; Lampen, N; de Harven, E; Clarkson, B D

    1975-09-01

    This report describes the surface architecture of leukemic cells, as seen by scanning electron microscopy in 34 patients with acute nonlymphoblastic leukemia. Six patients with myeloblastic, 4 with promyelocytic, 10 with myelomonocytic, 8 with monocytic, 4 with histiocytic and 2 with undifferentiated leukemia were studied. Under the scanning electron microscope most leukemia histiocytes and monocytes appeared similar and were characterized by the presence of large, well developed broad-based ruffled membranes or prominent raised ridge-like profiles, resembling ithis respect normal monocytes. Most cells from patients with acute promyelocytic or myeloblastic leukemia exhibited narrower ridge-like profiles whereas some showed ruffles or microvilli. Patients with myelomonocytic leukemia showed mixed populations of cells with ridge-like profiles and ruffled membranes whereas cells from two patients with undifferentiated leukemia had smooth surfaces, similar to those encountered in cells from patients with acute lymphoblastic leukemia. It appears that nonlymphoblastic and lymphoblastic leukemia cells (particularly histiocytes and monocytes) can frequently be distinquished on the basis of their surface architecture. The surface features of leukemic histiocytes and monocytes are similar, suggesting that they may belong to the same cell series. The monocytes seem to have characteristic surface features recognizable with the scanning electron microscope and differ from most cells from patients with acute granulocytic leukemia. Although overlap of surface features and misidentification can occur, scanning electron microscopy is a useful adjunct to other modes of microscopy in the study and diagnosis of acute leukemia.

  11. Does granulocyte colony-stimulating factor exacerbate radiation-induced acute lung injury in rats?

    International Nuclear Information System (INIS)

    Miura, Gouji; Awaya, Hitomi; Matsumoto, Tsuneo; Tanaka, Nobuyuki; Matsunaga, Naofumi

    2000-01-01

    Radiation pneumonitis (RP) frequently occurs as a complication of thoracic irradiation. However, the mechanism of RP is not well known. Activated neutrophils are a possible pathogenesis of RP. Neutrophil activation induced by granulocyte colony-stimulating factor (G-CSF) may exacerbate RP. We studied the effects of recombinant human G-CSF on acute lung injury induced by thoracic irradiation using rats. Animals were divided into three groups: sham irradiation with saline control, irradiation alone, and irradiation with G-CSF. Actual irradiation was given as a single fraction of 16 Gy delivered to the right hemithorax. G-CSF at a dose of 12 μg/body was administered subcutaneously once a day from 14 to 18 days after actual irradiation. Lung injury was evaluated 21 days after irradiation by bronchoalveolar lavage (BAL) fluid findings and the lung wet/dry weight (W/D) ratio. Neutrophil and lymphocyte counts in BAL fluid and the W/D ratio were significantly increased in the irradiation alone and the irradiation with G-CSF groups compared with those of the sham irradiation+saline control group. However, there was no significant difference observed between the irradiation alone and irradiation with G-CSF groups. In conclusion, this study suggests that postradiation administration of G-CSF does not exacerbate acute lung injury induced by thoracic irradiation in rats. (author)

  12. Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Mochizuki, D.Y.; Eisenman, J.R.; Conlon, P.J.; Park, L.S.; Urdal, D.L.

    1986-01-01

    The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1β, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum together with recombinant GM-CSF that had been radiolabeled with 125 I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis

  13. Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine

    International Nuclear Information System (INIS)

    Husain, Zaheed; Almeciga, Ingrid; Delgado, Julio C.; Clavijo, Olga P.; Castro, Januario E.; Belalcazar, Viviana; Pinto, Clara; Zuniga, Joaquin; Romero, Viviana; Yunis, Edmond J.

    2006-01-01

    Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 μM) in the presence of 0.1 mM H 2 O 2 demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis

  14. Expression of granulocyte colony-stimulating factor receptor correlates with prognosis in oral and mesopharyngeal carcinoma.

    Science.gov (United States)

    Tsuzuki, H; Fujieda, S; Sunaga, H; Noda, I; Saito, H

    1998-02-15

    Granulocyte colony-stimulating factor receptors (G-CSFRs) have been observed on the surface of not only hematopoietic cells but also several cancer cells. The stimulation of G-CSF has been demonstrated to induce proliferation and activation of G-CSFR-positive cells. In this study, we investigated the expression of G-CSFR on the surface of tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCC) by an immunohistochemical approach. Of 58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases (62.1%) were positive for G-CSFR and G-CSF, respectively. There was no association between G-CSFR expression and G-CSF staining. In the group positive for G-CSFR expression, relapse was significantly more likely after primary treatment (P = 0.0069), whereas there was no association between G-CSFR expression and age, sex, tumor size, lymph node metastasis, and clinical stage. Also, the G-CSFR-positive groups had a significantly lower disease-free and overall survival rate than the G-CSFR-negative groups (P = 0.0172 and 0.0188, respectively). However, none of the clinical markers correlated significantly with G-CSF staining, nor did the status of G-CSF production influence the overall survival. The results imply that assessment of G-CSFR may prove valuable in selecting patients with oral and mesopharyngeal SCC for aggressive therapy.

  15. Fluorine-18 fluorodeoxyglucose splenic uptake from extramedullary hematopoiesis after granulocyte colony-stimulating factor stimulation.

    Science.gov (United States)

    Abdel-Dayem, H M; Rosen, G; El-Zeftawy, H; Naddaf, S; Kumar, M; Atay, S; Cacavio, A

    1999-05-01

    Two patients with sarcoma, one with recurrent osteosarcoma of the spine and the other with metastatic synovial cell sarcoma, were treated with high-dose chemotherapy that produced severe leukopenia. The patients received granulocyte colony-stimulating factor (G-CSF) to stimulate the bone marrow (480 mg given subcutaneously twice daily for 5 to 7 days); their responses were seen as a marked increase in peripheral leukocyte count with no change in the erythrocyte or platelet counts. The patients had fluorine-18 fluorodeoxyglucose (F-18 FDG) imaging 24 hours after the end of G-CSF treatment. Diffusely increased uptake of F-18 FDG was seen in the bone marrow in both patients. In addition, markedly increased uptake in the spleen was noted in both, indicating that the spleen was the site of extramedullary hematopoiesis. The patients had no evidence of splenic metastases. The first patient had a history of irradiation to the dorsal spine, which was less responsive to G-CSF administration than was the nonirradiated lumbar spine.

  16. Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade.

    Science.gov (United States)

    Ouzounova, Maria; Lee, Eunmi; Piranlioglu, Raziye; El Andaloussi, Abdeljabar; Kolhe, Ravindra; Demirci, Mehmet F; Marasco, Daniela; Asm, Iskander; Chadli, Ahmed; Hassan, Khaled A; Thangaraju, Muthusamy; Zhou, Gang; Arbab, Ali S; Cowell, John K; Korkaya, Hasan

    2017-04-06

    It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced 'metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression.

  17. Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils

    International Nuclear Information System (INIS)

    Hanazono, Y.; Hosoi, T.; Kuwaki, T.; Matsuki, S.; Miyazono, K.; Miyagawa, K.; Takaku, F.

    1990-01-01

    We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism

  18. Indium-111 labeled purified granulocytes in the diagnosis of synthetic vascular graft infection

    International Nuclear Information System (INIS)

    Forstrom, L.A.; Dewanjee, M.K.; Chowdhury, S.; Brown, M.L.

    1988-01-01

    Indium-111 labeled leukocytes have been shown to be useful in the diagnosis of synthetic vascular graft infection. To minimize the potential effects of labeled red blood cells and platelets on image interpretation, the authors prepared purified autologous granulocytes (PG) from 84 ml of blood using Volex enhanced gravity sedimentation and Ficoll-Hypaque double density centrifugation. The labeling efficiency of PG with In-111 tropolone was 90 +/- 9% (mean +/- SD). Imaging was performed 18-24 hours following injection of approximately 445 microcuries of In-111 PG in 26 patients with suspected infection of vascular grafts that had been implanted 12 days to 12 years prior to the study. In ten patients with proven graft infection, seven had positive In-111 PG scans. Ten of 11 patients without infection had negative scans. In five patients with clinically equivocal findings, scan results were positive in one, negative in one, and equivocal in three. A false-positive scan occurred in a patient with an uninfected inflammatory pseudoaneurysm of an aortic graft. These results confirm an earlier report that In-111 PG imaging is a useful technique in the diagnosis of synthetic vascular graft infection

  19. Granulocyte Macrophage Colony-Stimulating Factor-Activated Eosinophils Promote Interleukin-23 Driven Chronic Colitis

    Science.gov (United States)

    Griseri, Thibault; Arnold, Isabelle C.; Pearson, Claire; Krausgruber, Thomas; Schiering, Chris; Franchini, Fanny; Schulthess, Julie; McKenzie, Brent S.; Crocker, Paul R.; Powrie, Fiona

    2015-01-01

    Summary The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target. PMID:26200014

  20. Does granulocyte colony-stimulating factor exacerbate radiation-induced acute lung injury in rats?

    Energy Technology Data Exchange (ETDEWEB)

    Miura, Gouji; Awaya, Hitomi; Matsumoto, Tsuneo; Tanaka, Nobuyuki; Matsunaga, Naofumi [Yamaguchi Univ., Ube (Japan). School of Medicine

    2000-08-01

    Radiation pneumonitis (RP) frequently occurs as a complication of thoracic irradiation. However, the mechanism of RP is not well known. Activated neutrophils are a possible pathogenesis of RP. Neutrophil activation induced by granulocyte colony-stimulating factor (G-CSF) may exacerbate RP. We studied the effects of recombinant human G-CSF on acute lung injury induced by thoracic irradiation using rats. Animals were divided into three groups: sham irradiation with saline control, irradiation alone, and irradiation with G-CSF. Actual irradiation was given as a single fraction of 16 Gy delivered to the right hemithorax. G-CSF at a dose of 12 {mu}g/body was administered subcutaneously once a day from 14 to 18 days after actual irradiation. Lung injury was evaluated 21 days after irradiation by bronchoalveolar lavage (BAL) fluid findings and the lung wet/dry weight (W/D) ratio. Neutrophil and lymphocyte counts in BAL fluid and the W/D ratio were significantly increased in the irradiation alone and the irradiation with G-CSF groups compared with those of the sham irradiation+saline control group. However, there was no significant difference observed between the irradiation alone and irradiation with G-CSF groups. In conclusion, this study suggests that postradiation administration of G-CSF does not exacerbate acute lung injury induced by thoracic irradiation in rats. (author)

  1. Effect of Temperature on Granulocyte and Monocyte Adsorption to Cellulose Acetate Beads.

    Science.gov (United States)

    Nishise, Shoichi; Takeda, Yuji; Abe, Yasuhiko; Sasaki, Yu; Nara, Hidetoshi; Asao, Hironobu; Ueno, Yoshiyuki

    2017-06-01

    Granulocyte and monocyte (GM) adsorptive apheresis (GMA) is an effective therapy for inflammatory disorders including inflammatory bowel disease (IBD). During GMA, the blood of a patient with IBD passes through a column to contact cellulose acetate (CA) beads at a temperature below body temperature, likely close to room temperature. Here we investigated the effect of temperature on GM adsorption to CA beads in vitro. We incubated peripheral blood with and without CA beads at 5°C, 25°C, 37°C, and 43°C and calculated the ratios of adsorbed GMs. The ratios of adsorbed GMs increased as the temperature was raised. Additionally, we measured complement activation fragment concentrations. C3a and C5a concentrations also increased as the temperature was raised, and C5a concentrations had a positive correlation with the ratios of adsorbed GMs. These results suggest that warming the column during GMA might increase GM adsorption to CA beads, thereby enhancing the clinical efficacy of GMA. © 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.

  2. Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

    International Nuclear Information System (INIS)

    Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake; Ozaki, Norio; Noda, Yukihiro

    2016-01-01

    Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100 μM) and doxorubicin (0.2 µM) decreased the cell survival rate, but olanzapine (1–100 µM) did not. Under granulocytic differentiation for 5 days, clozapine, even at a concentration of 25 μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H 4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H 4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H 4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H 4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation. - Highlights: • HL-60

  3. Involvement of the histamine H{sub 4} receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake [Division of Clinical Sciences and Neuropsychopharmacology, Faculty and Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 (Japan); Ozaki, Norio [Department of Psychiatry, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Noda, Yukihiro, E-mail: ynoda@meijo-u.ac.jp [Division of Clinical Sciences and Neuropsychopharmacology, Faculty and Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 (Japan)

    2016-09-01

    Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100 μM) and doxorubicin (0.2 µM) decreased the cell survival rate, but olanzapine (1–100 µM) did not. Under granulocytic differentiation for 5 days, clozapine, even at a concentration of 25 μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H{sub 4} receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H{sub 4} receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H{sub 4} receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H{sub 4} receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation

  4. HEMORRHAGIC-FEVER VIRUS-INFECTIONS IN AN ISOLATED RAIN-FOREST AREA OF CENTRAL LIBERIA - LIMITATIONS OF THE INDIRECT IMMUNOFLUORESCENCE SLIDE TEST FOR ANTIBODY SCREENING IN AFRICA

    NARCIS (Netherlands)

    van der Waals, F. W.; Pomeroy, K. L.; Goudsmit, J.; Asher, D. M.; Gajdusek, D. C.

    1986-01-01

    Serum samples from 119 healthy individuals and 106 epilepsy patients inhabiting Grand Bassa County, Liberia, were tested for antibodies to hemorrhagic fever viruses (HFV) by indirect immunofluorescence. E6 Vero cells infected with Lassa fever virus (LAS), Rift Valley Fever virus (RVF), Congo

  5. The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

    Science.gov (United States)

    Erlandsen, S L; Sherlock, L A; Bemrick, W J

    1990-04-01

    The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

  6. Longitudinal study of the indirect immunofluorescence and complement fixation tests for diagnosis of chagas' disease in immunosuppressed patients submitted to renal transplantation

    Directory of Open Access Journals (Sweden)

    José Fernando de Castro Figueiredo

    1993-12-01

    Full Text Available Clinical and serological follow-up of 7 patients submitted to renal transplantation and presenting positive serological reactions to Chagas 'disease before immunossupression did not show significant changes in indirect immunofluorescence and complement fixation titres for Chagas ' disease, or signs and symptoms indicating exacerbation of the disease during follow- up. In addition, 18 of 66 recipients of renal transplants considered to be non-chagasic before immunosuppression showed at least one positive result to the indirect immunofluorescence test for Chagas ' disease during the study period. The results suggest that the immunosuppression State induced in chagasic patients submitted to renal transplant did notpromoted exacerbation of the chronic infection in these patients and not interfere with the serological response of chronic chagasics, thus permitting the use of these serologic reactions for diagnostic purposes in these cases. However, the positive results ofthe indirect immunofluorescence test in non- chagasic patients indicate the needforjudicious interpretation ofthe indirect immunofluorescence test for the diagnosis of Chagas' disease in renal transplanted patients.

  7. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

    Directory of Open Access Journals (Sweden)

    Jonathan A Rose

    Full Text Available Pulmonary arterial hypertension (PAH is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  8. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Rose, Jonathan A; Wanner, Nicholas; Cheong, Hoi I; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  9. Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug-induced cell cycle perturbations in vitro

    DEFF Research Database (Denmark)

    Engelholm, S A; Spang-Thomsen, M; Vindeløv, L L

    1986-01-01

    A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small-cell carcinoma of the lung. Three tumors with different...... sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow...

  10. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

    Science.gov (United States)

    Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

    2012-12-21

    Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in

  11. Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

    Directory of Open Access Journals (Sweden)

    Sabine Lederer

    Full Text Available In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV, Puumala (PUUV, Seoul (SEOV, Saaremaa (SAAV, Dobrava (DOBV, Sin Nombre (SNV or Andes (ANDV. For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97; SEOV (China, n=5; DOBV (Romania, n=7; SNV (Canada, n=23; ANDV (Argentina and Chile, n=52. The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96% and/or IgM (n=131; 72%, while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99% of the patient sera were IgG and 131 (71% IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%. Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%. Of the 89 control sera, 2 (2% showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV.

  12. Green Approach To Synthesize Crystalline Nanoscale ZnII-Coordination Polymers: Cell Growth Inhibition and Immunofluorescence Study.

    Science.gov (United States)

    Mukherjee, Somali; Ganguly, Sumi; Manna, Krishnendu; Mondal, Sanchaita; Mahapatra, Supratim; Das, Debasis

    2018-04-02

    Five new coordination polymers (CPs) namely, [{Zn(μ 2 -H 2 O) 0.5 (5N 3 -IPA)(2,2'-bpe)}] ∞ (1), [{Zn(μ 2 -H 2 O) 0.5 (5N 3 -IPA)(1,10-phen)}] ∞ (2), [{Zn(5N 3 -IPA)(1,2-bpe)}] ∞ (3), [{Zn(5N 3 -IPA)(1,2-bpey)}] ∞ (4), and [{Zn(H 2 O)(5N 3 -IPA)(4,4'-tme)}(H 2 O) 0.5 ] ∞ (5) (5N 3 -H 2 IPA = 5-azidoisophthalic acid, 2,2'-bpe= 2,2'-bipyridine, 1,10-phen = 1,10-phenanthroline, 1,2-bpe = 1,2-bis(4-pyridyl)ethane, 1,2-bpey = 1,2-bis(4-pyridyl)ethylene, 4,4'-tme = 4,4'-trimethylenedipyridine), have been synthesized based on a mixed ligand approach adopting a solvothermal technique. Depending upon the intrinsic structural flexibility of the bis-pyridyl coligands, interesting structural topologies have also been observed in the resulting CPs: Sra SrAl2 type topology for 3 and a 3-fold interpenetrated dmp topology for 4. A green hand grinding technique has been implemented to reduce the particle size of the CPs to generate nanoscale CPs (NCPs). SEM studies of NCPs reveal the formation of square and spherical particles for NCP 1 and 2, respectively, and nano rod for NCP 3, 4, and 5. Remarkably, when scaled down to nano range all the NCPs retain their crystalline nature. The cytotoxic activity of the NCPs (1-5) has been studied using human colorectal carcinoma cells (HCT 116). Significant cell death is observed for NCP 2, which is further corroborated by cell growth inhibition study. The observed cell death is likely to be due to mitochondrial-assisted apoptosis as is evident from immunofluorescence study.

  13. Evaluation of a Direct Immunofluorescent Assay and/or Conjunctival Cytology for Detection of Canine Distemper Virus Antigen.

    Science.gov (United States)

    Athanasiou, Labrini V; Kantere, Maria C; Kyriakis, Constantinos S; Pardali, Dimitra; Adamama Moraitou, Katerina; Polizopoulou, Zoe S

    2018-04-01

    Canine distemper is a common and potentially lethal multisystemic disease caused by the Canine distemper virus (CDV). We evaluated the diagnostic performance of direct immunofluorescent assay (FA) and cytology to detect CDV antigen in conjunctival cells compared with an established polymerase chain reaction (PCR) detection assay used as a gold standard for CDV diagnosis. Samples were collected from 57 young dogs presenting with central nervous system signs compatible with distemper disease. Exfoliative epithelial cells were collected from the right and left conjunctiva of each animal using nylon-bristled cytobrushes for cytology and cotton swabs for FA and PCR. For the direct FA, samples were stained with anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate and imaged using a fluorescent microscope. Out of 57 dogs tested, 19 were PCR positive (15 positive in direct FA and 4 positive in cytology, including one that was negative by PCR), whereas 37 dogs were negative in all methods. A good agreement was observed between the FA and PCR, with a κ-value of 0.833 (95% CI: 0.678-0.989). Meanwhile, there was poor agreement between cytology and PCR (κ-value of 0.164; 95% CI: -0.045 to 0.373) and a fair agreement between FA and cytology (κ-value of 0.231; 95% CI: -0.026 to 0.487). Our results indicated a poor performance of cytology for the detection of CDV antigen. In contrast, FA is a 100% specific and an adequately sensitive assay (sensitivity: 78.95%, negative likelihood ratio: 0.21, 95% CI: 0.09-0.50) for antemortem diagnosis of canine distemper.

  14. Effects of human granulocyte-colony stimulating factor on fracture healing in rats

    International Nuclear Information System (INIS)

    Bozlar, M.; Aslan, B.; Kalaci, A.; Yanat, Ahmet N.; Baktiroglu, L.; Tasci, A.

    2005-01-01

    Granulocyte colony stimulation factor (G-CSF) is generally used to prevent and cure the neutropenia associated with chemotherapy and bone marrow transplantation. In addition to its effects on neutrophil function, G-CSF was found to have the characteristic of modulating the cytokines in the inflammatory response. Then, the question to answer is whether it has any effect on fracture healing and to what extent? In this study, we test the effects of G-CSF on the healing of tibia fracture in a rat model. This study was performed at Harran University, Sanliurfa, Turkey between July 2003 and August 2004. Twenty female, healthy Sprague-Dawley rats, weighing between 250 and 300 gm were divided into 2 groups, and their tibiae broken. The rats in the G-CSF group were injected subcutaneous with 25ug/kg/day of recombinant human G-CSF for 7 days, and the ones in the control group with 0.9% sodium chloride. Rats were sacrificed 3 weeks after surgery and then radiological, histological and biomechanical evaluations were performed. Biomechanical tests were performed at the Middle East Technical University, Ankara, Turkey.The median radiographic scores for the control group were calculated as 4.1, and 6.1 for the G-CSF group (p = 0.016). Cortex remodeling, callus formation, bone union and marrow changes values did not differ significantly (p > 0.05). Mechanical parameter (mean max-Load) values for the control group were found to be 24.0 +/- 3.0 N, and 241.5 +/-75.7 N for the G-CSF group (p 0.001). We found that G-CSF has an important effect on fracture healing. However, this effect requires further study. (author)

  15. Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

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    S Montagnani

    2009-12-01

    Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

  16. Time-dependent labelling course of human eosinophilic granulocytes after 3H thymidine application

    International Nuclear Information System (INIS)

    Walle, A.J.

    1975-01-01

    After intravenous injection of 0.1 μCi/g body weight 3 H-Thymidine and taking of blood samples in intervals of 6-12 hrs. on three test persons with healthy blood, the labelling course of the eosinophilic granulocytes was studied. The cells were classified in four groups, according to the relative frequency of the different degrees of labelling. The time-dependent labelling index curves showed a nawe-sheped course. Elimination of the eosinophilics from the blood is carried out according to the 'At-random'-principle. 12 hrs. p.i. already 10% of the eosinophilics in the blood were labelled with maximally 5 grains. The cell flow-in phase of 13 hrs. was succeeded by a flow-out phase of nearly the same duration, afthr the first labelling maximum of 17%. 80 hrs. p.i. the first massive in-flow of high-labelled cells containing more than 30 grains. After reaching the labelling maximum of 58%, the labelling index values decreased continuously. Until the 11th day p.i., appr. 50% of the eosinophilics were still labelled, after 17 days appr. 25%, more than 65% of which consisted of cells with only 2-4 grains. Comparison of the labelling index curves of the grain groups with each other shows at first a synchronous, then an increasingly asynchronous course, according to the desynchronization of the several eosinophilic generation cycles in the bone marrow which gets more significant in the course of time. (orig.) [de

  17. Isolated granulocytic sarcoma of the nasopharynx: a case report and review of the literature

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    Vishnu P

    2013-12-01

    Full Text Available Prakash Vishnu,1 Ravindra Reddy Chuda,2 Dick G Hwang,3 David M Aboulafia1,4 1Floyd and Delores Jones Cancer Institute at Virginia Mason Medical Center, Seattle, WA, USA; 2Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS, USA; 3Department of Pathology, Virginia Mason Medical Center, 4Division of Hematology, University of Washington, Seattle, WA, USA Abstract: Granulocytic sarcoma (GS is a rare extramedullary manifestation of acute myeloid leukemia (AML. It may also represent blastic transformation of myelodysplastic syndromes or myeloproliferative neoplasms. Although usually seen in the context of advanced and poorly controlled disease, it may also present as the first manifestation of illness, without concurrent bone marrow or blood involvement. In the medical literature, chloroma and GS are terms that have been used interchangeably with myeloid sarcoma. GS usually manifests as soft tissue or bony masses in several extracranial sites, such as bone, periosteum, and lymph nodes; involvement of the head and neck region is uncommon. We report a case of a woman with insidious onset of progressive nasal congestion and diminished hearing who was diagnosed with an isolated GS of the nasopharynx. With involved field radiotherapy, she achieved a complete remission of 12-months duration before being diagnosed with overt AML. She has remained disease-free for greater than 18 months following induction and consolidation chemotherapy. Through a MEDLINE®/PubMed® search we identified an additional 13 cases of nasopharyngeal GS. The median age was 37 years (range 1 to 81 years. The cases were equally distributed among the sexes. The most common presenting symptoms were conductive hearing loss and sinonasal congestion. Isolated GS was identified in six cases, and the median time from diagnosis of GS to AML was 12 months (range 3 to 48 months. The treatment varied, but responses were seen in all the patients who received

  18. Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms.

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    Haixiu Guo

    Full Text Available Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs. In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL. CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET and 5.3% of cases with primary myelofibrosis (PMF. Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR. Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F.

  19. Granulocyte colony-stimulating factor in repeated IVF failure, a randomized trial.

    Science.gov (United States)

    Aleyasin, Ashraf; Abediasl, Zhila; Nazari, Atefeh; Sheikh, Mahdi

    2016-06-01

    Recent studies have revealed key roles for granulocyte colony-stimulating factor (GCSF) in embryo implantation process and maintenance of pregnancy, and some studies showed promising results by using local intrauterine infusion of GCSF in patients undergoing in vitro fertilization (IVF). This multicenter, randomized, controlled trial included 112 infertile women with repeated IVF failure to evaluate the efficacy of systemic single-dose subcutaneous GCSF administration on IVF success in these women. In this study, the Long Protocol of ovarian stimulation was used for all participants. Sealed, numbered envelopes assigned 56 patients to receive subcutaneous 300 µg GCSF before implantation and 56 in the control group. The implantation (number of gestational sacs on the total number of transferred embryos), chemical pregnancy (positive serum β-HCG), and clinical pregnancy (gestational sac and fetal heart) rates were compared between the two groups. This trial is registered at www.irct.ir (IRCT201503119568N11). The successful implantation (18% vs 7.2%, P=0.007), chemical pregnancy (44.6% vs 19.6%, P=0.005), and clinical pregnancy (37.5% vs 14.3%, P=0.005) rates were significantly higher in the intervention group than in the control group. After adjustment for participants' age, endometrial thickness, good-quality oocyte counts, number of transferred embryos, and anti-Mullerian hormone levels, GCSF treatment remained significantly associated with successful implantation (OR=2.63, 95% CI=1.09-6.96), having chemical pregnancy (OR= 2.74, 95% CI=1.11-7.38) and clinical pregnancy (OR=2.94, 95% CI=1.23-8.33). In conclusion, administration of single-dose systemic subcutaneous GCSF before implantation significantly increases the IVF success, implantation, and pregnancy rates in infertile women with repeated IVF failure. © 2016 Society for Reproduction and Fertility.

  20. Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis

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    Giniatullina Raisa

    2011-06-01

    Full Text Available Abstract Background Granulocyte colony stimulating factor (GCSF is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS. ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. Methods Human mutant G93A superoxide dismutase (SOD1 ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. Results Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. Conclusions GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.

  1. Granulocyte colony-stimulating factor mobilizes dormant hematopoietic stem cells without proliferation in mice.

    Science.gov (United States)

    Bernitz, Jeffrey M; Daniel, Michael G; Fstkchyan, Yesai S; Moore, Kateri

    2017-04-06

    Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41 Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment. © 2017 by The American Society of Hematology.

  2. Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep.

    Science.gov (United States)

    Regan, Sheena L P; McFarlane, James R; O'Shea, Tim; Andronicos, Nicholas; Arfuso, Frank; Dharmarajan, Arun; Almahbobi, Ghanim

    2015-08-01

    The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations. © 2015 Society for Reproduction and Fertility.

  3. Correlation Between Serum Concentrations of N-Desmethylclozapine and Granulocyte Levels in Patients with Schizophrenia: A Retrospective Observational Study.

    Science.gov (United States)

    Smith, Robert L; Haslemo, Tore; Andreassen, Ole A; Eliasson, Erik; Dahl, Marja-Liisa; Spigset, Olav; Molden, Espen

    2017-11-01

    Clozapine is restricted to use in patients with treatment-refractory schizophrenia due to the risk of a serious drop in absolute neutrophil granulocyte count (ANC). The formation of reactive, unstable metabolites (adducts) has been suggested as a mechanism of clozapine-induced granulocyte decline. These adducts are not detectable in vivo, but stable clozapine metabolites could potentially be indirect pharmacokinetic measures of adduct formation. The present retrospective observational study investigated the correlation between concentrations of N-desmethylclozapine, the major stable clozapine metabolite, and ANC in a real-life population of clozapine-treated patients. Patients were included from a therapeutic drug monitoring service at the Center for Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway, between March 2005 and December 2015. Information about clozapine and N-desmethylclozapine steady-state trough concentrations, as well as accompanying measurements of ANC, were collected from the laboratory database. Correlations of serum concentrations of N-desmethylclozapine and clozapine (and their respective ratios) with ANC were investigated by linear mixed-model analysis. Overall, 129 patients with 855 measurements of clozapine/N-desmethylclozapine concentrations and ANC (range 0.9-19 × 10 9 cells/L, median 4.6) were included. Concentrations of N-desmethylclozapine, but not clozapine, correlated significantly and positively with ANC (estimated model slope 0.0011 × 10 9 cells/L/nM; p = 0.002), and the N-desmethylclozapine/clozapine ratio also positively correlated with ANC (p = 0.040). N-Desmethylclozapine level and ANC significantly correlated in this real-life population of schizophrenia patients. The positive correlation, which was also present for the metabolic ratio, might reflect reduced clozapine availability for the formation of reactive metabolites potentially affecting granulocyte level. However, as our findings were based on ANC mainly

  4. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

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    Cindy Franklin

    Full Text Available Chronic graft-versus-host disease (cGVHD is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

  5. Early Expansion of Circulating Granulocytic Myeloid-derived Suppressor Cells Predicts Development of Nosocomial Infections in Patients with Sepsis.

    Science.gov (United States)

    Uhel, Fabrice; Azzaoui, Imane; Grégoire, Murielle; Pangault, Céline; Dulong, Joelle; Tadié, Jean-Marc; Gacouin, Arnaud; Camus, Christophe; Cynober, Luc; Fest, Thierry; Le Tulzo, Yves; Roussel, Mikael; Tarte, Karin

    2017-08-01

    Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression, but their clinical impact remains poorly defined. To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in patients with sepsis. Peripheral blood transcriptomic analysis was performed on 29 patients with sepsis and 15 healthy donors. A second cohort of 94 consecutive patients with sepsis, 11 severity-matched intensive care patients, and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8, and ARG1, were up-regulated in the peripheral blood of patients with sepsis. CD14 pos HLA-DR low/neg monocytic (M)-MDSCs were expanded in intensive care unit patients with and without sepsis and CD14 neg CD15 pos low-density granulocytes/granulocytic (G)-MDSCs were more specifically expanded in patients with sepsis (P sepsis. G-MDSCs, made of immature and mature granulocytes expressing high levels of degranulation markers, were specifically responsible for arginase 1 activity. High initial levels of G-MDSCs, arginase 1, and S100A12 but not M-MDSCs were associated with subsequent occurrence of nosocomial infections. M-MDSCs and G-MDSCs strongly contribute to T-cell dysfunction in patients with sepsis. More specifically, G-MDSCs producing arginase 1 are associated with a higher incidence of nosocomial infections and seem to be major actors of sepsis-induced immune suppression.

  6. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

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    Chan Koon H

    2010-09-01

    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  7. [Application of Immunohistochemistry and Immunofluorescence Staining in Detection of Phospholipase A2 Receptor on Paraffin Section of Renal Biopsy Tissue].

    Science.gov (United States)

    Dong, Hong-rui; Wang, Yan-yan; Wang, Guo-qin; Sun, Li-jun; Cheng, Hong; Chen, Yi-pu

    2015-10-01

    immunohistochemical staining and immunofluorescence staining, the preferred method of antigen repair is water bath heat repair plus trypsin repair.

  8. Granulocyte-colony stimulating factor (G-CSF improves motor recovery in the rat impactor model for spinal cord injury.

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    Tanjew Dittgen

    Full Text Available Granulocyte-colony stimulating factor (G-CSF improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. Previously we have employed the mouse hemisection SCI model and studied motor function after subcutaneous or transgenic delivery of the protein. To further broaden confidence in animal efficacy data we sought to determine efficacy in a different model and a different species. Here we investigated the effects of G-CSF in Wistar rats using the New York University Impactor. In this model, corroborating our previous data, rats treated subcutaneously with G-CSF over 2 weeks show significant improvement of motor function.

  9. Clinical outcome after stem cell mobilization with granulocyte-colony-stimulating factor after acute ST-elevation myocardial infarction:

    DEFF Research Database (Denmark)

    Ripa, Rasmus S; Jørgensen, Erik; Kastrup, Jens

    2013-01-01

    Background. Granulocyte-colony-stimulating factor (G-CSF) has been investigated in trials aiming to promote recovery of myocardial function after myocardial infarction. Long-term safety-data have never been reported. A few studies indicated an increased risk of in-stent re-stenosis. We aimed to i.......8; 0.3). Conclusions. We found no indication of increased risk of adverse events up to 5 years after G-CSF treatment. These results support the continued investigation of G-CSF for cardiac therapy....

  10. Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

    Science.gov (United States)

    Dunham, S P; Onions, D E

    2001-06-21

    A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

  11. Ontogeny of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the beagle.

    Science.gov (United States)

    Nothdurft, W; Braasch, E; Calvo, W; Prümmer, O; Carbonell, F; Grilli, G; Fliedner, T M

    1984-04-01

    The pattern of development of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the course of canine ontogeny was studied by means of the agar culture technique. Colony formation was stimulated by colony stimulating activity (CSA) in serum from lethally irradiated dogs in combination with erythrocyte-depleted peripheral blood leukocytes from normal adult dogs. The colonies thus obtained in cultures from the different organs were in general large (estimated maximum 50 000 cells) and consisted predominantly of mononucleated macrophages, suggesting that, in these studies, a progenitor cell with high proliferative potential (HPP-CFC) has been monitored. In the yolk sac, a transitory GM-CFC pool became established between day 23 and day 48 of gestation, reaching maximum numbers of approximately 41 X 10(3) per organ on days 36/37. At the same time the GM-CFC concentration in blood collected from the heart also reached a maximum of about 31 X 10(3)/ml, indicating its carrier function for the migration of GM-CFC. In the liver a quasi-exponential increase in the GM-CFC numbers took place between days 36/37 and days 57 to 59 when a total of about 15.2 X 10(6) was found but thereafter and up to day 4 post partum the GM-CFC numbers decreased by almost two orders of magnitude. A continuous increase in the GM-CFC numbers was found in the spleen between day 42 of gestation and day 4 post partum when a maximum of 5.1 X 10(6) to 8.7 X 10(6) was reached. In contrast to the GM-CFC numbers in the liver, the splenic GM-CFC dropped only by 50% of peak values when the dogs reached adulthood. The bone marrow always had the highest incidence of GM-CFC, the concentration per 10(6) cells being 18.7 X 10(3)/10(6) cells on days 45/46, the earliest time point at which cultures could be set up. The absolute GM-CFC numbers in the two femora increased continuously between days 45/46 and day 4 post partum in parallel with the growth of the bones. In the thymus a relatively small

  12. Bone marrow immunoscintigraphy using technetium-99m anti-granulocyte antibody in multiple myeloma

    International Nuclear Information System (INIS)

    Sohn, Sang-Kyun; Kim, Dong-Hwan; Park, So-Hyang; Ahn, Byeong-Cheol; Lee, Sang-Woo; Chun, Kyung-Ah; Kim, Jung-Gyun; Lee, Kyu Bo; Lee, Jaetae; Song, Hong-Suk

    2002-01-01

    Conventional skeletal radiography and bone scan have certain limitations in the initial evaluation of bone and bone marrow lesions in multiple myeloma (MM). In this study we investigated the value of bone marrow immunoscintigraphy (BMIS) using anti-granulocyte monoclonal antibody (AGA) for the diagnosis of bone involvement of MM, in comparison with bone scan and skeletal radiography. Whole-body BMIS using technetium-99m-labelled AGA was performed in 22 MM patients (15 male, 7 female) and the imaging findings compared with those of skeletal radiography and 99m Tc-methylene diphosphonate bone scan. The findings of bone marrow aspiration and serum biochemical findings were also compared with BMIS findings. Abnormal findings of BMIS were defined as presence of a focal photon defect in the axial skeleton or expansion of peripheral bone marrow. A total of 124 focal lesions were detected in 19 subjects (86%) by skeletal radiography, bone scan or BMIS. BMIS detected 92 lesions (74%) in 19 subjects, whereas skeletal radiography detected 58 focal lesions (47%) in 14 and bone scan 40 lesions (32%) in 11. Fifty-one (41%) of the 124 lesions were only seen on BMIS. Spine and pelvic lesions were better visualised by BMIS, whereas skull lesions were better seen with skeletal radiography, and bone scan detected more lesions in the ribs. Marrow expansion was noted in 15 subjects (68%) on BMIS, and its grade correlated with marrow cellularity and myeloma cell percentage in bone marrow aspirates (P=0.0055 and P=0.0541, respectively). BMIS revealed abnormal lesions in one of three stage II patients and 17 out of 19 stage III patients. The number of lesions of the thoracolumbar vertebrae on BMIS was correlated with cellularity (P=0.0393), but not with myeloma cell percentage (P=0.1262). These findings suggest that the results of BMIS with 99m Tc-labelled AGA correlate with clinical stage, and thus reflect the functional status of bone marrow in MM patients. BMIS might be useful for the

  13. Regulation of wound healing by granulocyte-macrophage colony-stimulating factor after vocal fold injury.

    Directory of Open Access Journals (Sweden)

    Jae-Yol Lim

    Full Text Available OBJECTIVES: Vocal fold (VF scarring remains a therapeutic challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF facilitates epithelial wound healing, and recently, growth factor therapy has been applied to promote tissue repair. This study was undertaken to investigate the effect of GM-CSF on VF wound healing in vivo and in vitro. METHODS: VF scarring was induced in New Zealand white rabbits by direct injury. Immediately thereafter, either GM-CSF or PBS was injected into the VFs of rabbits. Endoscopic, histopathological, immunohistochemical, and biomechanical evaluations of VFs were performed at 3 months post-injury. Human vocal fold fibroblasts (hVFFs were cultured with GM-CSF. Production of type I and III collagen was examined immunocytochemically, and the synthesis of elastin and hyaluronic acids was evaluated by ELISA. The mRNA levels of genes related to ECM components and ECM production-related growth factors, such as HGF and TGF-ß1, were examined by real time RT-PCR. RESULTS: The GM-CSF-treated VFs showed reduced collagen deposition in comparison to the PBS-injected controls (P<0.05. Immunohistochemical staining revealed lower amounts of type I collagen and fibronectin in the GM-CSF-treated VFs (P<0.05 and P<0.01, respectively. Viscous and elastic shear moduli of VF samples were significantly lower in the GM-CSF group than in the PBS-injected group (P<0.001 and P<0.01, respectively. Mucosal waves in the GM-CSF group showed significant improvement when compared to the PBS group (P = 0.0446. GM-CSF inhibited TGF-β1-induced collagen synthesis by hVFFs (P<0.05 and the production of hyaluronic acids increased at 72 hours post-treatment (P<0.05. The expressions of HAS-2, tropoelastin, MMP-1, HGF, and c-Met mRNA were significantly increased by GM-CSF, although at different time points (P<0.05. CONCLUSION: The present study shows that GM-CSF offers therapeutic potential for the remodeling of VF wounds and the promotion of VF

  14. Diagnosis, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: A Review.

    Science.gov (United States)

    Sanchez, Edgar; Vannier, Edouard; Wormser, Gary P; Hu, Linden T

    2016-04-26

    Lyme disease, human granulocytic anaplasmosis (HGA), and babesiosis are emerging tick-borne infections. To provide an update on diagnosis, treatment, and prevention of tick-borne infections. Search of PubMed and Scopus for articles on diagnosis, treatment, and prevention of tick-borne infections published in English from January 2005 through December 2015. The search yielded 3550 articles for diagnosis and treatment and 752 articles for prevention. Of these articles, 361 were reviewed in depth. Evidence supports the use of US Food and Drug Administration-approved serologic tests, such as an enzyme immunoassay (EIA), followed by Western blot testing, to diagnose extracutaneous manifestations of Lyme disease. Microscopy and polymerase chain reaction assay of blood specimens are used to diagnose active HGA and babesiosis. The efficacy of oral doxycycline, amoxicillin, and cefuroxime axetil for treating Lyme disease has been established in multiple trials. Ceftriaxone is recommended when parenteral antibiotic therapy is recommended. Multiple trials have shown efficacy for a 10-day course of oral doxycycline for treatment of erythema migrans and for a 14-day course for treatment of early neurologic Lyme disease in ambulatory patients. Evidence indicates that a 10-day course of oral doxycycline is effective for HGA and that a 7- to 10-day course of azithromycin plus atovaquone is effective for mild babesiosis. Based on multiple case reports, a 7- to 10-day course of clindamycin plus quinine is often used to treat severe babesiosis. A recent study supports a minimum of 6 weeks of antibiotics for highly immunocompromised patients with babesiosis, with no parasites detected on blood smear for at least the final 2 weeks of treatment. Evidence is evolving regarding the diagnosis, treatment, and prevention of Lyme disease, HGA, and babesiosis. Recent evidence supports treating patients with erythema migrans for no longer than 10 days when doxycycline is used and prescription

  15. Comparison of two strategies for the treatment of radiogenic leukopenia using granulocyte colony stimulating factor

    International Nuclear Information System (INIS)

    Adamietz, I.A.; Rosskopf, B.; Dapper, F.D.; Lieven, H. von; Boettcher, H.D.

    1996-01-01

    Purpose: Radiation-induced leukopenia can cause a delay or discontinuation of radiotherapy. This complication can be overcome with the use of granulocyte colony-stimulating factor (G-CSF). However, an uncertainty exists regarding the mode of application of G-CSF in patients treated with radiotherapy. For this reason, the efficacy of two strategies for the administration of G-CSF in irradiated patients was compared in a prospective randomized clinical study. Methods and Materials: Forty-one patients who developed leukopenia ( 9 per liter) while undergoing radiotherapy were treated with G-CSF at a daily dose of 5 μg/kg. The first group received single injections of G-CSF as required (n = 21). The second group received G-CSF on at least 3 consecutive days (n = 20). An analysis was made of the changes in leucocyte counts, the number of days on which radiotherapy had to be interrupted, and the side effects of growth-factor treatment. Results: An increase in leucocyte values in the peripheral blood was observed in all patients treated with G-CSF. In the group which received G-CSF when required, two injections (range: 1-8) were administered in most cases. In the second group, most of the patients received three injections (range: 3-9). The average duration of therapy interruptions due to leukopenia was 4.8 days (0-28) in the first therapy arm and 2.5 (0-20) in the second arm. The variance in the duration of therapy interruptions between the two groups was not significant (p = 0.2). Radiotherapy had to be terminated in two patients due to thrombocytopenia but the application of G-CSF did not seem to be a reason of decreasing platelet counts. Conclusions: Our results reveal that G-CSF is safe and effective in the treatment of radiation-induced leukopenia regardless of the mode of application. Because the calculated difference related to radiation treatment interruptions has no clinical relevance, both approaches examined in our study appear reasonable.

  16. CXCL1 can be regulated by IL-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Roy Monica

    2012-01-01

    Full Text Available Abstract Background Granulocytes generally exert protective roles in the central nervous system (CNS, but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE, the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined. Methods CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and in situ hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques. Results CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS or pertussis toxin (PTX, the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease. Conclusions This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.

  17. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. II. - X irradiation effects and influence of hyperthermia on the radiosensitivity

    International Nuclear Information System (INIS)

    Bueren, J.A.; Nieto, M.

    1983-01-01

    The effects of the X-irradiation on the viability of the granulocyte-macrophage precursors, has been determined by means of the agar diffusion chamber culture technique. The results show the high radiosensitivity of these cells, with survival parameter similar to those previously reported in the literature about different granulocyte-macrophage precursors. When a hyperthermic treatment is performed prior to the X-irradiation, a radiosensitization phenomenon is observed due to the synergism existent between hyperthermia and X rays on the lethality of the precursors. (Authors) 37 refs

  18. Automated analysis of flow cytometric data for measuring neutrophil CD64 expression using a multi-instrument compatible probability state model.

    Science.gov (United States)

    Wong, Linda; Hill, Beth L; Hunsberger, Benjamin C; Bagwell, C Bruce; Curtis, Adam D; Davis, Bruce H

    2015-01-01

    Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of state modeling (PSM). Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000. Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher

  19. Comparative strain analysis of Anaplasma phagocytophilum infection and clinical outcomes in a canine model of granulocytic anaplasmosis.

    Science.gov (United States)

    Scorpio, Diana G; Dumler, J Stephen; Barat, Nicole C; Cook, Judith A; Barat, Christopher E; Stillman, Brett A; DeBisceglie, Kristen C; Beall, Melissa J; Chandrashekar, Ramaswamy

    2011-03-01

    A pilot study was conducted to determine whether existing human or canine strains of Anaplasma phagocytophilum would reproduce clinical disease in experimentally inoculated dogs similar to dogs with naturally acquired granulocytic anaplasmosis. Six hounds were inoculated intravenously with one human and two canine strains of A. phagocytophilum that were propagated in vitro in HL-60 cells or in infected autologous neutrophils. Infected dogs were monitored for lethargy, anorexia, petechiae, lymphadenopathy, and fever. Dogs were assessed for complete blood count (CBC), serum chemistry, and serology (IFA and SNAP® 4Dx®); for A. phagocytophilum blood load by quantitative polymerase chain reaction; and for cytokine production. Prominent clinical signs were generalized lymphadenopathy and scleral injection; only one dog developed fever lasting 4 days. Notable laboratory alterations included sustained leukopenia and thrombocytopenia in all dogs. A. phagocytophilum morulae were noted in blood between days 10 and 11, although all dogs retained A. phagocytophilum DNA in blood through day 60. All dogs seroconverted by days 10-15 by IFA, and by days 17-30 by SNAP 4Dx; cytokine analyses revealed 10-fold increases in interleukin-2 and interleukin-18 in the neutrophil-propagated 98E4 strain-infected dog. All A. phagocytophilum strains produced infection, although canine 98E4 strain reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis when recognized clinically. Therefore, this strain should be considered for use in future studies of A. phagocytophilum canine infection models.

  20. Successful treatment of fusarium solani ecthyma gangrenosum in a patient affected by leukocyte adhesion deficiency type 1 with granulocytes transfusions

    Directory of Open Access Journals (Sweden)

    Hassen Assia

    2010-10-01

    Full Text Available Abstract Background Ecthyma gangrenosum (EG manifests as a skin lesion affecting patients suffering extreme neutropenia and is commonly associated with Pseudomonas aeruginosa in immunocompromised patients. Leukocyte adhesion deficiency I (LAD I which count among primary immunodeficiency syndromes of the innate immunity, is an autosomal recessive disorder characterized in its severe phenotype by a complete defect in CD18 expression on neutrophils, delayed cord separation, chronic skin ulcers mainly due to recurrent bacterial and fungal infections, leucocytosis with high numbers of circulating neutrophils and an accumulation of abnormally low number of neutrophils at sites of infection. Case Presentation We report at our knowledge the first case of a child affected by LAD-1, who experienced during her disease course a multi-bacterial and fungal EG lesion caused by fusarium solani. Despite targeted antibiotics and anti-fungi therapy, the lesion extended for as long as 18 months and only massive granulocytes pockets transfusions in association with G-CSF had the capacity to cure this lesion. Conclusion We propose that granulocytes pockets transfusions will be beneficial to heal EG especially in severely immunocompromised patients.

  1. Infliximab- and Immunosuppressant-Resistant Crohn’s Disease Successfully Treated with Adsorptive Granulocyte Apheresis Combined with Prednisolone

    Directory of Open Access Journals (Sweden)

    Munenori Itagaki

    2012-02-01

    Full Text Available Activated granulocytes, monocytes, and platelets appear to be closely involved in active Crohn’s disease (CD. Adsorptive granulocyte apheresis (GCAP is a new treatment for inflammatory bowel disease. GCAP was used to treat a 23-year-old female patient with CD resistant to both infliximab (IFX and azathioprine (AZA. At 16 years of age, the patient underwent a partial ileal resection for peritonitis caused by perforative ileitis. On pathological examination of the resected specimen, the diagnosis was CD. Mesalazine was started, but the patient did not comply with therapy. She was admitted to our hospital again in 2007 due to an acute exacerbation. IFX induction therapy was started. The combination of both AZA daily and IFX every 8 weeks was continued as maintenance therapy. However, she developed severe abdominal pain in September 2009. Computed tomography revealed ileitis and ascending colitis, and blood tests showed high inflammatory response marker levels. She was considered to have IFX- and AZA-resistant CD. Initial intravenous steroid therapy did not result in any improvement. Therefore, weekly GCAP therapy was given for 5 weeks, which immediately improved the inflammatory response markers. GCAP combined with prednisolone could be effective for IFX- and AZA-refractory CD.

  2. Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

    Science.gov (United States)

    Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

    2018-01-01

    The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  3. Japanese Society for Laboratory Hematology flow cytometric reference method of determining the differential leukocyte count: external quality assurance using fresh blood samples.

    Science.gov (United States)

    Kawai, Y; Nagai, Y; Ogawa, E; Kondo, H

    2017-04-01

    To provide target values for the manufacturers' survey of the Japanese Society for Laboratory Hematology (JSLH), accurate standard data from healthy volunteers were needed for the five-part differential leukocyte count. To obtain such data, JSLH required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (JSLH-Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (ICSH-Diff) and the manual differential count obtained by microscopy (Manual-Diff). First, the reference laboratory performed an imprecision study of JSLH-Diff and ICSH-Diff, as well as performing comparison among JSLH-Diff, Manual-Diff, and ICSH-Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of JSLH-Diff, Manual-Diff, and ICSH-Diff. Simultaneously, six manufacturers' laboratories provided their own representative values by using automated hematology analyzers. The precision of both JSLH-Diff and ICSH-Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the JSLH-Diff, ICSH-Diff, and Manual-Diff methods conformed to the criteria. When the imprecision and accuracy of JSLH-Diff were assessed at seven laboratories, the CV% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of CD45 positive leukocytes were identified as normal leukocytes by JSLH-Diff. When JSLH-Diff method were validated by comparison with Manual-Diff and ICSH-Diff, JSLH-Diff showed good performance as a reference method. © 2016 John Wiley & Sons Ltd.

  4. Flow Cytometric DNA Analysis Using Cytokeratin Labeling for Identification of Tumor Cells in Carcinomas of the Breast and the Female Genital Tract

    Directory of Open Access Journals (Sweden)

    Rainer Kimmig

    2001-01-01

    Full Text Available Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

  5. The effect of granulocyte-colony stimulating factor on rotator cuff healing after injury and repair.

    Science.gov (United States)

    Ross, David; Maerz, Tristan; Kurdziel, Michael; Hein, Joel; Doshi, Shashin; Bedi, Asheesh; Anderson, Kyle; Baker, Kevin

    2015-05-01

    The failure rate of tendon-bone healing after repair of rotator cuff tears remains high. A variety of biologic- and cell-based therapies aimed at improving rotator cuff healing have been investigated, and stem cell-based techniques have become increasingly more common. However, most studies have focused on the implantation of exogenous cells, which introduces higher risk and cost. We aimed to improve rotator cuff healing by inducing endogenous stem cell mobilization with systemic administration of granulocyte-colony stimulating factor (G-CSF). We asked: (1) Does G-CSF administration increase local cellularity after acute rotator cuff repair? (2) Is there histologic evidence that G-CSF improved organization at the healing enthesis? (3) Does G-CSF administration improve biomechanical properties of the healing supraspinatus tendon-bone complex? (4) Are there micro-MRI-based observations indicating G-CSF-augmented tendon-bone healing? After creation of full-thickness supraspinatus tendon defects with immediate repair, 52 rats were randomized to control or G-CSF-treated groups. G-CSF was administered for 5 days after repair and rats were euthanized at 12 or 19 postoperative days. Shoulders were subjected to micro-MR imaging, stress relaxation, and load-to-failure as well as blinded histologic and histomorphometric analyses. G-CSF-treated animals had significantly higher cellularity composite scores at 12 and 19 days compared with both control (12 days: 7.40 ± 1.14 [confidence interval {CI}, 5.98-8.81] versus 4.50 ± 0.57 [CI, 3.58-5.41], p = 0.038; 19 days: 8.00 ± 1.00 [CI, 6.75-9.24] versus 5.40 ± 0.89 [CI, 4.28-6.51], p = 0.023) and normal animals (12 days: p = 0.029; 19 days: p = 0.019). There was no significant difference between G-CSF-treated animals or control animals in ultimate stress (MPa) and strain, modulus (MPa), or yield stress (MPa) and strain at either 12 days (p = 1.000, p = 0.104, p = 1.000, p = 0.909, and p = 0.483, respectively) or 19 days (p = 0

  6. Primary granulocyte colony-stimulating factor prophylaxis during the first two cycles only or throughout all chemotherapy cycles in patients with breast cancer at risk for febrile neutropenia

    NARCIS (Netherlands)

    Aarts, M.J.; Peters, F.P.; Mandigers, C.M.P.W.; Dercksen, M.W.; Stouthard, J.M.; Nortier, H.J.; Laarhoven, H.W.M. van; Warmerdam, L.J. van; Wouw, A.J. van de; Jacobs, E.M.G.; Mattijssen, V.; Rijt, C.C. van der; Smilde, T.J.; Velden, A.W. van der; Temizkan, M.; Batman, E.; Muller, E.W.; Gastel, S.M. van; Borm, G.F.; Tjan-Heijnen, V.C.

    2013-01-01

    PURPOSE: Early breast cancer is commonly treated with anthracyclines and taxanes. However, combining these drugs increases the risk of myelotoxicity and may require granulocyte colony-stimulating factor (G-CSF) support. The highest incidence of febrile neutropenia (FN) and largest benefit of G-CSF

  7. Primary Granulocyte Colony-Stimulating Factor Prophylaxis During the First Two Cycles Only or Throughout All Chemotherapy Cycles in Patients With Breast Cancer at Risk for Febrile Neutropenia

    NARCIS (Netherlands)

    Aarts, Maureen J.; Peters, Frank P.; Mandigers, Caroline M.; Dercksen, M. Wouter; Stouthard, Jacqueline M.; Nortier, Hans J.; van Laarhoven, Hanneke W.; van Warmerdam, Laurence J.; van de Wouw, Agnes J.; Jacobs, Esther M.; Mattijssen, Vera; van der Rijt, Carin C.; Smilde, Tineke J.; van der Velden, Annette W.; Temizkan, Mehmet; Batman, Erdogan; Muller, Erik W.; van Gastel, Saskia M.; Borm, George F.; Tjan-Heijnen, Vivianne C. G.

    2013-01-01

    Purpose Early breast cancer is commonly treated with anthracyclines and taxanes. However, combining these drugs increases the risk of myelotoxicity and may require granulocyte colony-stimulating factor (G-CSF) support. The highest incidence of febrile neutropenia (FN) and largest benefit of G-CSF

  8. Dose intensity of standard adjuvant CMF with granulocyte colony-stimulating factor for premenopausal patients with node-positive breast cancer

    NARCIS (Netherlands)

    deGraaf, H; Willemse, PHB; Bong, SB; Piersma, H; Tjabbes, T; vanVeelen, H; Coenen, JLLM; deVries, EGE

    1996-01-01

    The effects of granulocyte colony-stimulating factor (G-CSF) on total dose and dose intensity of standard oral adjuvant CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) chemotherapy were studied in premenopausal patients with node-positive breast cancer. Treatment consisted of standard CMF

  9. Effects of recombinant human granulocyte colony-stimulating factor on leucopenia in zidovudine-treated patients with AIDS and AIDS related complex, a phase I/II study

    NARCIS (Netherlands)

    van der Wouw, P. A.; van Leeuwen, R.; van Oers, R. H.; Lange, J. M.; Danner, S. A.

    1991-01-01

    Twelve male patients, eight with the acquired immunodeficiency syndrome (AIDS) and four with AIDS related complex (ARC), who had zidovudine associated neutropenia (less than 1 x 10(9) neutrophils/l) were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) in a phase I/II

  10. Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

    Science.gov (United States)

    Ribeiro-Filho, Antonio Carlos; Buri, Marcus Vinicius; Barros, Carlos Castilho; Dreyfuss, Juliana Luporini; Nader, Helena Bonciani; Justo, Giselle Zenker; Craveiro, Rogério Bastos; Pesquero, João Bosco; Miranda, Antonio; Ferreira, Alice Teixeira; Paredes-Gamero, Edgar Julian

    2016-07-07

    All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

  11. Granulocyte colony-stimulating factor in glycogen storage disease type 1b. Results of the European Study on Glycogen Storage Disease Type 1

    NARCIS (Netherlands)

    Visser, G.; Rake, J.P.; Labrune, P.; Leonard, J.V.; Moses, S.; Ullrich, K.; Wendel, U.; Groenier, K.H.; Smit, G.P.

    2002-01-01

    Patients with glycogen storage disease type 1b (GSD-1b) have neutropenia and neutrophil dysfunction that predispose to frequent infections and inflammatory bowel disease (IBD), for which granulocyte colony-stimulating factor (GCSF) is given. To investigate the use and the value of GCSF treatment in

  12. Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Kabaya, Koji; Watanabe, Masahiko; Kusaka, Masaru; Seki, Masatoshi (Kirin Brewery Co., Ltd., Gunma (Japan). Pharmaceutical Research Laboratory); Fushiki, Masato

    1994-08-01

    The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 [mu]g/body/day from day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also reveals an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation. (author).

  13. Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice

    International Nuclear Information System (INIS)

    Kabaya, Koji; Watanabe, Masahiko; Kusaka, Masaru; Seki, Masatoshi; Fushiki, Masato.

    1994-01-01

    The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 μg/body/day from day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also reveals an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation. (author)

  14. Ovarian granulocytic sarcoma: a case report and magnetic resonance imaging findings; Sarcoma granulocitico no ovario: relato de caso e achados na ressonancia magnetica

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Licia Pacheco [Hospital Geral de Fortaleza (HGF), CE (Brazil). Servico de Diagnostico por Imagem], e-mail: licia_p@hotmail.com; Silveira, Claudio Regis Sampaio [Hospital Geral de Fortaleza (HGF), CE (Brazil). Servico de Radiologia; Costa, Fabricio da Silva [Universidade Estadual do Ceara (UECE), CE (Brazil); Monte, Hipolito [Hospital Monte Klinikum, Fortaleza, CE (Brazil)

    2008-12-15

    Granulocytic sarcoma (chloroma) is a tumor consisting of myeloid precursors in an extramedullary site. It is complication of both acute and chronic myelogenous leukemias. Although the lesion can occur at any site, ovarian involvement is rare. We report a case of ovary tumor associated with acute myeloid leukaemia and its imaging appearance on magnetic resonance. (author)

  15. Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils

    Science.gov (United States)

    The use of immunomodulators is a promising area for biotherapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease, particularly during periods of peak disease incidence. Cytokines, including granulocyte colony-stimulating factor (G-CSF), are one class of compounds that...

  16. Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes.

    Science.gov (United States)

    Prodjinotho, Ulrich F; von Horn, Charlotte; Debrah, Alex Y; Batsa Debrah, Linda; Albers, Anna; Layland, Laura E; Hoerauf, Achim; Adjobimey, Tomabu

    2017-07-01

    Helminth parasites are known to be efficient modulators of their host's immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on

  17. Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

    Science.gov (United States)

    Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

    2018-02-01

    Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

  18. Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways

    DEFF Research Database (Denmark)

    Vestergaard, Maj Linea; Awan, Aashir; Warzecha, Caroline Becker

    2016-01-01

    onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance......This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging...

  19. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

    Energy Technology Data Exchange (ETDEWEB)

    Birch, C J; Lehmann, N I; Hawker, A J; Marshall, J A; Gust, I D [Fairfield Hospital for Communicable Diseases, Victoria (Australia). Virology Dept.

    1979-07-01

    Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.

  20. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

    Energy Technology Data Exchange (ETDEWEB)

    Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

    1982-12-01

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

  1. Dew drops on spider web appearance: a newly named pattern of IgG4 deposition in pemphigus with direct immunofluorescence

    Directory of Open Access Journals (Sweden)

    Marian Dmochowski

    2017-08-01

    Full Text Available Novel appearances in cutaneous pathology as well as mucocutaneous clinical signs are being described which indicate that this is still an attractive area for exploration. The H + E histology terms of “decorated tomb stoning” and “undecorated tomb stoning”, advocated by some pathologists, are misleading and as such should be avoided. Here, an appearance of IgG4 pemphigus deposits examined cost-effectively with direct immunofluorescence and suggested to be called “dew drops on spider web” is depicted in depth.

  2. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

    International Nuclear Information System (INIS)

    Birch, C.J.; Lehmann, N.I.; Hawker, A.J.; Marshall, J.A.; Gust, I.D.

    1979-01-01

    Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy. (author)

  3. Effect of intravenous plasma transfusion on granulocyte and monocyte oxidative and phagocytic activity in dairy calves with failure of passive immunity.

    Science.gov (United States)

    Yang, Victoria C; Rayburn, Maire C; Chigerwe, Munashe

    2017-12-01

    Plasma administration has been recommended in calves older than 48h with failure of passive immunity (FPI) to provide immunity consistent with adequate colostral ingestion. However, the protective serum immunoglobulin G (IgG) concentrations (≥1000mg/dL) of plasma derived IgG only lasts up to 12h. In addition to IgG, maternally derived colostral cells also confer immunity. The objective of the study was to determine the effect of intravenous plasma transfusion on granulocyte and monocyte oxidative and phagocytic activity in calves with FPI. Twenty-seven, one day-old, Jersey calves were assigned into 3 groups. The colostral (CL, N=9) group received 3L of colostrum once by oroesophageal tubing. Two other groups of calves received 1L of colostrum once by oroesophageal tubing and were assigned based on their health status (sick or non-sick) at 4days of age, as the sick-group (SG, N=7) or the non-sick (NG, N=11) groups. At 4days of age, the SG and NG groups were administered plasma intravenously at 30mL/kg. Granulocyte and monocyte oxidative and phagocytic activity was determined by flow cytometry. There was no significant difference in the granulocyte and monocyte oxidative or phagocytic activity among the 3 groups (P>0.05). Plasma administration had no significant effect on the oxidative or phagocytic activity of granulocytes or monocytes. In clinical practice, plasma administration for enhancing oxidative or phagocytic activity of granulocytes or monocytes, alone, might not be justified in calves with FPI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Application of microchip CGE for the analysis of PEG-modified recombinant human granulocyte-colony stimulating factors.

    Science.gov (United States)

    Park, Eun Ji; Lee, Kyung Soo; Lee, Kang Choon; Na, Dong Hee

    2010-11-01

    The purpose of this study was to evaluate the microchip CGE (MCGE) for the analysis of PEG-modified granulocyte-colony stimulating factor (PEG-G-CSF) prepared with PEG-aldehydes. The unmodified and PEG-modified G-CSFs were analyzed by Protein 80 and 230 Labchips on the Agilent 2100 Bioanalyzer. The MCGE allowed size-based separation and quantitation of PEG-G-CSF. The Protein 80 Labchip was useful for PEG-5K-G-CSF, while the Protein 230 Labchip was more suitable for PEG-20K-G-CSF. The MCGE was also used to monitor a search for optimal PEG-modification (PEGylation) conditions to produce mono-PEG-G-CSF. This study demonstrates the usefulness of MCGE for monitoring and optimizing the PEGylation of G-CSF with the advantages of speed, minimal sample consumption, and automatic quantitation.

  5. Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

    DEFF Research Database (Denmark)

    Boje, Alex; Moesby, Lise; Timm, Michael

    2012-01-01

    The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed...... that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest...... concentration of testosterone (120 µM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further...

  6. Septal endocarditis, bone infection and severe leg ischemia detected in Tc-99m labelled monoclonal anti granulocyte scan

    International Nuclear Information System (INIS)

    Bechelaghem, A.I; Habbeche, M; Benlabgaa, R; Ghedbane, IE; Hanzal, A; Khelifa, A; Mechcken, F; Bourezak, SE; Bouyoucef, SE

    2006-01-01

    Patient 28 years old has continued to have a persistent fever (39.2 O C), despite ten days treatment by specific antibiotics for bacterial endocarditis associated to a recent claudication of the right lower leg. The persistent fever has motivated a 99mTc-labelled monoclonal anti granulocyte scan which has showed an important uptake in the myocardial septum, and other infection locations in temporal bone and in right tibial arteries. Two days after, a nanocolloids-99mTc WBS showed no uptake in the heart area, a total absence of uptake of the nanocolloids in the bone marrow of right tibia b and cranial SPECT views confirmed the infectious site in the right temporal bone. New antibiotic strategy was adopted successfully associated with surgical amputation of the right lower leg (au)

  7. FEATURES OF CHEMILUMINESCENT ACTIVITY OF NEUTROPHILIC GRANULOCYTES IN PATIENTS WITH CHRONIC GASTRITIS, CHRONIC ATROPHIC GASTRITIS AND GASTRIC CANCER

    Directory of Open Access Journals (Sweden)

    O. V. Smirnova

    2017-01-01

    Full Text Available Chronic gastritis is the most common disease of gastro-intestinal tract. Precancerous potential is among most important epidemiological features of chronic gastritis. Immune system plays a distinct role in transformation from precancerous state to malignancy. In this context, the aim of our work was a study of spontaneous and induced chemiluminescence activity of neutrophilic granulocytes in patients with chronic superficial gastritis, chronic atrophic gastritis and gastric cancer. The work presents results of comprehensive laboratory examination of patients with chronic gastritis (CG (a total of 85 persons. 25 patients with chronic atrophic gastritis (CAG, and 50 patients with gastric cancer (GC at the age of 19 to 70 years were enrolled. Control group included 115 healthy donors without gastrointestinal complaints at the age of 19 to 67 years. The study was performed with venous blood samples taken from cubital vein into Vacutainer tubes with sodium heparin (5 U/mL prior to starting any pathogenic treatment. Evaluation of spontaneous and induced chemiluminescence was performed for 90 minutes at a 36-channel “CL 3606” chemiluminescence analyzer (Russia. In our study, patients with gastric cancer showed clear unidirectional changes in chemiluminescent activity of neutrophilic granulocytes (NG. When measuring spontaneous and induced NG chemiluminescence, we diagnosed a decreased phagocytic activity characterized by prolonged time-to-peak and area under the curve for spontaneous and induced CL, thus presuming longer activation time required in cases of reduced phagocytic function. The NG activity in patients with chronic gastritis is not impaired, but, similar changes of time-to-peak and area under were detected. Chemiluminescent activity of NG is increased in the group of CAG patients, and, considering similar changes in activation time and area under the curve, NG also produce greater amount of reactive oxygen species. Thus, for all H

  8. Influence of neutrophile granulocyte/lymphocyte ratio (NLR on poor prognosis of elderly AECOPD patients during hospital stay

    Directory of Open Access Journals (Sweden)

    Jian-Rong Cui

    2016-01-01

    Full Text Available Objective: To discuss the influence of neutrophile granulocyte/lymphocyte ratio(NLR to the poor prognosis of elderly AECOPD patients during the stay in hospital. Method: A total of 133 cases elderly patients with AECOPD admitted in our hospital from March 2013 to September 2014 were selected, and divided them into death group (31 cases and survival group (102 cases according to in-hospital death occurrence; To compare the on admission general clinical data, therapy method, lung function, blood routine examination [white blood cell count (WBC, neutrophile granulocyte/lymphocyte ratio(NLR], C-reactive protein (CRP, blood gas analysis and blood biochemical indexes in both groups, and drew ROC curve for a analysis of the clinical value of NLR in the prediction of death. Results: Among 133 cases of elderly AECOPD patients: the proportion of combined pulmonary heart disease and mechanical ventilation in death group was higher than that in survival group, PaCO2, WBC count, neutrophil count, NLR, CRP level in death group was higher, but lymphocyte count, serum albumin(ALB in death group was lower; multiple logistic regression analysis showed that NLR presented independent positive correlation with the in-hospital death in elderly AECOPD patients; ROC curve analysis showed that the ROCAUC of NLR to the inhospital death in elderly AECOPD patients was 0.787, the best diagnostic node value was 7.3, sensitivity and specificity were 77.4% and 74.5% respectively; bounded by NLR(7.3, divided patients into NLR≥7.3 group and NLR<7.3 group, hospital stays, CRP level and mortality in NLR≥7.3 group were higher than that in NLR<7.3 group. Conclusion: NLR was the high risk factor of the in-hospital death in elderly AECOPD patients, early detection of NLR level had a certain difference to the evaluation for short-term prognosis of elderly AECOPD patients and guide treatment.

  9. Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

    Science.gov (United States)

    Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

    2016-01-01

    Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

  10. Construction of iron-polymer-graphene nanocomposites with low nonspecific adsorption and strong quenching ability for competitive immunofluorescent detection of biomarkers in GM crops.

    Science.gov (United States)

    Yin, Kaifei; Liu, Anran; Shangguan, Li; Mi, Li; Liu, Xu; Liu, Yuanjian; Zhao, Yuewu; Li, Ying; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2017-04-15

    We developed a new immunofluorescent biosensor by utilizing a novel nanobody (Nb) and iron-polymer-graphene nanocomposites for sensitive detection of 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacdterium tumefaciens strain CP4 (CP4-EPSPS), which considered as biomarkers of genetically modified (GM) crops. Specifically, we prepared iron doped polyacrylic hydrazide modified reduced graphene nanocomposites (Fe@RGO/PAH) by in-situ polymerization approach and subsequent a one-pot reaction with hydrazine. The resulting Fe@RGO/PAH nanocomposites displayed low nonspecific adsorption to analytes (11% quenching caused by nonspecific adsorption) due to electrostatic, energetic and steric effect of the nanocomposites. After Nb immobilizing, the as-prepared Fe@RGO/PAH/Nbs showed good selectivity and high quenching ability (92% quenching) in the presence of antigen (Ag) and polyethylene glycol (PEG) modified CdTe QDs (Ag/QDs@PEG), which is a nearly 4 fold than that of the unmodified GO in same condition. The high quenching ability of Fe@RGO/PAH/Nbs can be used for detection of CP4-EPSPS based on competitive immunoassay with a linearly proportional concentration range of 5-100ng/mL and a detection limit of 0.34ng/mL. The good stability, reproducibility and specificity of the resulting immunofluorescent biosensor are demonstrated and might open a new window for investigation of fluorescent sensing with numerous multifunctional graphene based materials. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants

    Directory of Open Access Journals (Sweden)

    JAIME A. TEIXEIRA DA SILVA

    2015-05-01

    Full Text Available Abstract. Teixeira da Silva JA. 2015. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants. Nusantara Bioscience 7: 26-32. Syngonium podophyllum L. (arrowhead vine is a popular leafy indoor pot plant whose tissue culture has been established, primarily through in vitro shoot culture, but several interesting aspects have not yet been explored. In this study, cv. ‘White Butterfly’ was used to investigate the response of shoot formation to alternative gelling agents and media additives. Gellan gum (Gelrite® at 2 g/L resulted in greater leaf production, plantlet fresh weight and higher chlorophyll content (SPAD value than all other gelling agents tested, including agar, Bacto agar, phytagel, oatmeal agar, potato dextrose agar, barley starch and corn starch, when on a basal Hyponex® (NPK = 6.5: 6: 19; 3 g/L medium. Several alternative liquid medium additives tested (low and full fat milk, Coca-Cola®, coffee, Japanese green, Oolong and Darjeeling teas negatively impacted plant growth, stunted roots and decreased chlorophyll content (SPAD value of leaves. Plant growth on medium with refined sucrose or table sugar responded similarly. Poor growth was observed when crude extract from a high rebaudioside-containing stevia (Stevia rebaudiana Bertoni line - an artificial sweetener - was used. Leaf tissue from the control did not show any endopolyploidy but low levels of endopolyploidy (8C were detected in some treatments.

  12. Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

    International Nuclear Information System (INIS)

    Baldock, Daniel; Nocker, Andreas; Nebe-von-Caron, Gerhard; Bongaerts, Roy

    2013-01-01

    Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR ® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR ® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pK a value. (paper)

  13. A Fast, Easy, and Customizable Eight-Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse.

    Science.gov (United States)

    Daubeuf, François; Becker, Julien; Aguilar-Pimentel, Juan Antonio; Ebel, Claudine; Hrabě de Angelis, Martin; Hérault, Yann; Frossard, Nelly

    2017-06-19

    The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  14. Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling

    International Nuclear Information System (INIS)

    Machherndl-Spandl, S; Suessner, S; Danzer, M; Proell, J; Gabriel, C; Lauf, J; Sylie, R; Klein, H-U; Béné, M C; Weltermann, A; Bettelheim, P

    2013-01-01

    Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34 + ) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34 + progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository

  15. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes; Analise por citometria de fluxo da expressao da proteina p21 em linfocitos humanos irradiados

    Energy Technology Data Exchange (ETDEWEB)

    Santos, N.F.G.; Amaral, A., E-mail: neyliane@gmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Energia Nuclear. Laboratorio de Modelagem e Biodosimetria Aplicada; Freitas-Silva, R. [Universidade Federal de Pernambuco (UFPE), Garanhuns, PE (Brazil). Departamento de Ciencias Naturais e Exatas; Pereira, V.R.A. [Fundacao Oswaldo Cruz (FIOCRUZ), Recife, PE (Brazil). Centro de Pesquisas Aggeu Magalhaes. Departamento de Imunologia. Lab. de Imunoparasitologia; Tasat, D.R. [Universidad Nacional de General San Martin, Buenos Aires (Argentina). Escuela de Ciencia y Tecnologia. Laboratorio de Biologia Celular del Pulmon

    2013-08-15

    Cell cycle blockage in G1 is a mechanism p21 protein-regulated and coupled to DNA damage response to permit genetic content analysis, damage repair and cell death. Analysis of proteins that participates of this response has progressed with new analytic tools, and data contributes to comprehension of radioinduced molecular events as well as to new approaches on practices that employ ionizing radiation. On this perspective, the aim of this research was to evaluate, by flow cytometry, p21 expression on irradiated human lymphocytes, maintained under different experimental conditions. Peripheral blood samples from 10 healthy subjects were irradiated with doses of 0 (non-irradiated), 1, 2 and 4 Gy. Lymphocytes were processed to analysis on ex vivo (no cultured) condition and after 24; 48 and 72 hours culture, with and without phytohemagglutinin stimulation. p21 protein expression levels were measured by flow cytometry, as percentage values. Results indicate that flow cytometric assay allows detection of changes on p21 expression, since it was detected significant increase on phytohemagglutinin-stimulated samples, for all times, against basal expression (ex vivo). However, it was not observed significant alterations on p21 protein radioinduced levels, for all doses, times and culture conditions analyzed. These results not indicate so p21 protein as bioindicator of ionizing radiation exposure. Nevertheless, data confirmation may to require analysis of a more numerous population. (author)

  16. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

    Science.gov (United States)

    Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

    2007-01-01

    Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

  17. Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Vigre, Håkan; Sørensen, Vibeke

    2005-01-01

    by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracelluaris exposed pigs with or without current proliferative enteropathy. (c) 2004......The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when...... later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive...

  18. Reações de imunofluorescência indireta: algumas modificações de sua técnica Indirect immunofluorescence tests: some modifications of their technique

    Directory of Open Access Journals (Sweden)

    Cláudio Santos Ferreira

    1973-09-01

    Full Text Available Propõem-se modificações de procedimentos rotineiros de reações de imunofluorescência indireta. A eluição das amostras de sangue dessecado contidas em papel-filtro é feita em placas ("godets" com tampas. Alças de níquel-cromo substituem pipetas capilares para a transferência de amostras de eluatos para as lâminas de microscopia. Propõe-se uma técnica rápida e eficiente de lavagem das preparações.Some simplifications intended for routine operations included immunofluorescence tests are proposed. The elution of dried blood samples is performed on test plates (water-color mixing plates, with a lid instead of in test tubes. Nickel-chromium wire loops are substituted for capillary pipettes in transferring eluate samples to microscope slides. A rapid and efficient washing technique is proposed.

  19. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico

    1995-06-01

    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

  20. Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces

    Directory of Open Access Journals (Sweden)

    Souza Doris Sobral Marques

    2003-01-01

    Full Text Available In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA, was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

  1. Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

    KAUST Repository

    Sugumar, Thennarasu; Ganesan, Pugalenthi; Harishankar, Murugesan; Dhinakar Raj, Gopal

    2013-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

  2. Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

    KAUST Repository

    Sugumar, Thennarasu

    2013-06-25

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

  3. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gornowicz-Porowska, Justyna; Seraszek-Jaros, Agnieszka; Bowszyc-Dmochowska, Monika; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Bartkiewicz, Paweł; Dmochowski, Marian

    2017-02-01

    Pemphigus and bullous pemphigoid (BP) are identified by autoantibodies (abs) against desmoglein 1, 3 (DSG1/3) and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF) using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection) and modified (IgG4 detection) mosaic for indirect immunofluorescence (IIFc - IIF commercial, IIFm - IIF modified) and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP) molecular diagnostics. To compare diagnostic accuracy of commercial (IgG detection) and modified (IgG4 detection) mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher's exact test. There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1)/desmoglein3 (DSG3)/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

  4. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays

    Directory of Open Access Journals (Sweden)

    Justyna Gornowicz-Porowska

    2017-02-01

    Full Text Available Introduction : Pemphigus and bullous pemphigoid (BP are identified by autoantibodies (abs against desmoglein 1, 3 (DSG1/3 and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection and modified (IgG4 detection mosaic for indirect immunofluorescence (IIFc – IIF commercial, IIFm – IIF modified and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP molecular diagnostics. Aim : To compare diagnostic accuracy of commercial (IgG detection and modified (IgG4 detection mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Material and methods : Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher’s exact test. Results: There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1/desmoglein3 (DSG3/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. Conclusions : The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

  5. Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

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    Honglei Chen

    2012-10-01

    Full Text Available Caveolin-1 (Cav-1 expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV-associated GC (EBVaGC is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029 that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032 and was positively associated with Cav-1 expression (r = 0.432, p < 0.001. EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

  6. A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM).

    Science.gov (United States)

    Pessina, Augusto; Croera, Cristina; Bayo, Maria; Malerba, Ilaria; Passardi, Laura; Cavicchini, Loredana; Neri, Maria G; Gribaldo, Laura

    2004-03-01

    In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a

  7. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I.-Development of the in vivo culture and effects induced by the hyperthermia

    International Nuclear Information System (INIS)

    Bueren, J. A.; Nieto, M.

    1983-01-01

    The present report shows the agar diffusion chamber technique for culturing granulocyte- macrophage precursor cells, obtained from mice bone marrow. Diffusion chambers containing the bone marrow suspension are implanted intraperitoneally Into mice and constitute a compartment which avoids the migration of cells, but allows the transit of the mouse biological fluxes, necessary for the cellular proliferation. By means of this technique, we studied the lethal effects of the hyperthermia on the precursors and their capacity to repair sublethal damage. (Author) 129 refs

  8. Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

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    James A Cotton

    Full Text Available Giardia duodenalis (syn. G. intestinalis, G. lamblia is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time

  9. Selective engraftment of the granulocyte compartment after allogeneic bone marrow transplantation in a patient with severe aplastic anemia.

    Science.gov (United States)

    Barriga, F J; Legues, M E; Bertin, P

    1996-05-01

    We present a patient with severe aplastic anemia who had partial engraftment with full chimerism after allogeneic bone marrow transplantation from an HLA identical sibling. A 3-year-old girl with severe aplastic anemia (SAA) received a bone marrow transplantation (BMT) from an HLA identical brother 9 months after her diagnosis. Before BMT she was red blood cell tranfusion dependent, had an absolute neutrophil count (ANC) of 1,000-1,500 x 10(9)/1 and a platelet count of 15-19,000 x 10(9)/1. She was conditioned with 800 cGy total body irradiation (TBI) and cyclophosphamide and received 3X10(8) nucleated cells/kg. She reached an ANC of 1500 x 10(9)/1 on day +35 but her reticulocyte and platelet counts did not recover. A bone marrow aspirate and biopsy post BMT showed hypoplasia with marked decrease in megakaryocyte and red blood cell precursors. The granulocyte compartment showed a left shift with predominance of promyelocytes and myelocytes. The karyotype showed full chimerism (46,XY) with no 46,XX metaphases. This case illustrates the possibility of a bone marrow microenvironment defect as the cause of SAA.

  10. Effect of granulocyte colony stimulating factor (G-CSF on IVF outcomes in infertile women: An RCT

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    Maryam Eftekhar

    2016-05-01

    Full Text Available Background: Despite major advances in assisted reproductive techniques, the implantation rates remain relatively low. Some studies have demonstrated that intrauterine infusion of granulocyte colony stimulating factor (G-CSF improves implantation in infertile women. Objective: To assess the G-CSF effects on IVF outcomes in women with normal endometrial thickness. Materials and methods: In this randomized controlled clinical trial, 100 infertile women with normal endometrial thickness who were candidate for IVF were evaluated in two groups. Exclusion criteria were positive history of repeated implantation failure (RIF, endocrine disorders, severe endometriosis, congenital or acquired uterine anomaly and contraindication for G-CSF (renal disease, sickle cell disease, or malignancy. In G-CSF group (n=50, 300 μg trans cervical intrauterine of G-CSF was administered at the oocyte retrieval day. Controls (n=50 were treated with standard protocol. Chemical, clinical and ongoing pregnancy rates, implantation rate, and miscarriage rate were compared between groups. Results: Number of total and mature oocytes (MII, two pronuclei (2PN, total embryos, transferred embryos, quality of transferred embryos, and fertilization rate did not differ significantly between two groups. So there were no significant differences between groups in chemical, clinical and ongoing pregnancy rate, implantation rate, and miscarriage rate Conclusion: our result showed in normal IVF patients with normal endometrial thickness, the intrauterine infusion of G-CSF did not improve pregnancy outcomes.

  11. Granulocyte Macrophage Colony Stimulating Factor Supplementation in Culture Media for Subfertile Women Undergoing Assisted Reproduction Technologies: A Systematic Review

    Science.gov (United States)

    Siristatidis, Charalampos; Vogiatzi, Paraskevi; Salamalekis, George; Creatsa, Maria; Vrachnis, Nikos; Glujovsky, Demián; Iliodromiti, Zoe; Chrelias, Charalampos

    2013-01-01

    Granulocyte macrophage colony stimulating factor (GM-CSF) is a cytokine/growth factor produced by epithelial cells that exerts embryotrophic effects during the early stages of embryo development. We performed a systematic review, and six studies that were performed in humans undergoing assisted reproduction technologies (ART) were located. We wanted to evaluate if embryo culture media supplementation with GM-CSF could improve success rates. As the type of studies and the outcome parameters investigated were heterogeneous, we decided not to perform a meta-analysis. Most of them had a trend favoring the supplementation with GM-CSF, when outcomes were measured in terms of increased percentage of good-quality embryos reaching the blastocyst stage, improved hatching initiation and number of cells in the blastocyst, and reduction of cell death. However, no statistically significant differences were found in implantation and pregnancy rates in all apart from one large multicenter trial, which reported favorable outcomes, in terms of implantation and live birth rates. We propose properly conducted and adequately powered randomized controlled trials (RCTs) to further validate and extrapolate the current findings with the live birth rate to be the primary outcome measure. PMID:23509457

  12. Combined application of alginate dressing and human granulocyte-macrophage colony stimulating factor promotes healing in refractory chronic skin ulcers.

    Science.gov (United States)

    Huang, Guobao; Sun, Tangqing; Zhang, Lei; Wu, Qiuhe; Zhang, Keyan; Tian, Qingfen; Huo, Ran

    2014-06-01

    The aim of the present study was to evaluate the clinical therapeutic effect of the combined application of alginate and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on the healing of refractory chronic skin ulcers. A single center, three arm, randomized study was performed at Jinan Central Hospital (Jinan, Shandong, China). A total of 60 patients with refractory chronic skin ulcers, which persisted for >1 month, were enrolled and randomly assigned into one of the following three groups: alginate dressing/rhGM-CSF group (group A), rhGM-CSF only group (group B) and conventional (vaseline dressing) group (group C). The wound area rate was measured, granulation and color were observed and pain was evaluated. The data were summarized and statistical analysis was performed. The results demonstrated that group A exhibited a significantly faster wound healing rate and lower pain score compared with the other groups (PCSF for the treatment of refractory chronic skin ulcers demonstrated significant advantages. It promoted the growth of granulation tissue, accelerated re-epithelialization and also effectively reduced wound pain, and thus improved the quality of life for the patient. This suggests that the combined application of alginate and rhGM-CSF may be an effective therapeutic strategy for the clinical treatment of refractory chronic skin ulcers.

  13. Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

    Science.gov (United States)

    Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

    2010-03-01

    The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

  14. The safety and clinical efficacy of recombinant human granulocyte colony stimulating factor injection for colon cancer patients undergoing chemotherapy

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    Jie Chen

    Full Text Available Summary Objective: The present study was designed to evaluate safety and efficacy of recombinant human granulocyte colony stimulating factor (G-CSF injection and whether this regimen could reduce the incidence of adverse events caused by chemotherapy. Method: A total of 100 patients with colon cancer who were treated with chemotherapy in our hospital from January 2011 to December 2014 were randomly divided into two groups, with 50 patients in each group. The patients in the treatment group received G-CSF 24 hours after chemotherapy for consecutive three days; the patients in the control group received the same dose of normal saline. Routine blood tests were performed 7 days and 14 days after chemotherapy. Results: Compared with the control group, the incidences of febrile neutropenia and leukocytopenia in the treatment group were significantly lower (p<0.05. In addition, the incidence of liver dysfunction in the treatment group was lower than that of the control group, without statistical significance. The incidence of myalgia in the treatment was higher than that of the control group without statistical significance. Conclusion: The present study indicated that G-CSF injection after chemotherapy is safe and effective for preventing adverse events in colon cancer patients with chemotherapy.

  15. The optimal use of granulocyte macrophage colony stimulating factor in radiation induced mucositis in head and neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Patni Nidhi

    2005-01-01

    Full Text Available Objective: Evaluation of response of granulocyte macrophage colony stimulating factor (GM-CSF on acute radiation toxicity profile in head and neck squamous cell carcinoma. Methods and Materials: Thirty three patients with proven stage I or II head & neck carcinoma received conventional external beam radiation therapy. Out of these, six patients received postoperative adjuvant therapy while remaining 27 received definitive RT. Patients were given 100 mcg GM-CSF subcutaneously per day along with radiation after they developed grade 2 mucositis and /or grade 2 dysphagia and / or complained of moderate pain. GM-CSF was administered till there was a subjective relief or objective response. Patients were evaluated for oral ulceration, swallowing status, pain and weight loss. Response to the treatment and patient outcome was assessed. Results: There was a decreased severity of mucositis and dysphagia in the evaluated patients. None of the patients suffered severe pain or required opioids. The mean weight loss was only 1.94%. Minimal side effects were experienced with GM-CSF. Conclusions: GM-CSF reduces the severity of acute side effects of radiation therapy thereby allowing completion of the treatment without interruption. Its remarkable response needs to be evaluated further in large randomized trials. The time of initiation and cessation of GM-CSF during radiation therapy and the required dose needs to be established.

  16. Role of FDG PET/CT in Diagnostic Evaluation of Granulocytic Sarcomas: A Series of 12 Patients.

    Science.gov (United States)

    Chandra, Piyush; Dhake, Sanket; Purandare, Nilendu; Agrawal, Archi; Shah, Sneha; Rangarajan, Venkatesh

    2017-01-01

    Granulocytic sarcoma (GS) is a rare extramedullary manifestation in patients with acute myeloid leukemia (AML), which can precede the diagnosis or occur in the posttreatment setting. Unlike its established role in other hematological malignancies like Hodgkin's on non-Hodgkin's disease, the exact role of positron emission tomography/computed tomography (PET/CT) in AML with or without GS remains to be defined. We retrospectively reviewed PET/CT scans of 12 patients with histologically proven GS. Marrow examination of these patients identified nine patients with isolated GS (without existent leukemia) and three patients with coexistent leukemia. PET/CT accurately identified all clinically evident GS in all 12 patients at initial staging and at follow-up with tumors, showing moderate to high 2-deoxy-2-fluoro-D-glucose uptake. Coexistent marrow disease was seen on PET/CT in three patients, which was confirmed on histopathology. In the same patients, PET/CT also detected additional sites of extramedullary disease in 66.6% (n = 8), which was either clinically occult or not evident on routine CT. PET/CT appears to be a highly sensitive imaging modality in diagnostic evaluation of GS. The most important indication of using PET/CT in these cases is to identify additional sites of clinically occult extramedullary disease, which can potentially impact treatment decisions and outcomes.

  17. IL-8 Expression in Granulocytic Epithelial Lesions of Idiopathic Duct-centric Pancreatitis (Type 2 Autoimmune Pancreatitis).

    Science.gov (United States)

    Ku, Yuna; Hong, Seung-Mo; Fujikura, Kohei; Kim, Sung Joo; Akita, Masayuki; Abe-Suzuki, Shiho; Shiomi, Hideyuki; Masuda, Atsuhiro; Itoh, Tomoo; Azuma, Takeshi; Kim, Myung-Hwan; Zen, Yoh

    2017-08-01

    Type 2 autoimmune pancreatitis (type 2 AIP) develops in isolation or sometimes in association with ulcerative colitis. Its diagnosis requires the histologic confirmation of granulocytic epithelial lesions (GELs) with no diagnostic biomarker currently available. This study aimed to elucidate the tissue expression of cytokines and their diagnostic value in this condition. In quantitative polymerase chain reaction for multiple cytokines using tissue-derived mRNA, the expression level of interleukin (IL)-8 was markedly higher in type 2 AIP than in type 1 AIP (Ppancreatitis adjacent to pancreatic cancers (peritumoral pancreatitis) exhibited IL-8 expression in the epithelium (3/12; 25%) and inflammatory cells (10/12; 83%), expression levels were significantly lower than those in type 2 AIP (Ppancreatitis with 92% sensitivity and 92% to 100% specificity. Furthermore, CD3/IL-8-coexpressing lymphocytes were almost restricted to type 2 AIP. Interestingly, a similar pattern of IL-8 expression was also observed in colonic biopsies of ulcerative colitis. In conclusion, the overexpression of IL-8 may underlie the development of GELs in type 2 AIP, and IL-8 immunostaining or IL-8/CD3 double staining may become an ancillary method for its diagnosis. The similar expression pattern of IL-8 in ulcerative colitis also suggests a pathogenetic link between the 2 conditions.

  18. Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

    Science.gov (United States)

    Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

    2015-01-30

    Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells

    International Nuclear Information System (INIS)

    Moonen, P.; Mermod, J.J.; Ernst, J.F.; Hirschi, M.; DeLamarter, J.F.

    1987-01-01

    Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein-lacking any carbohydrate- had by far the highest specific activity observed for the recombinant hGM-CSFs

  20. Granulocyte colony stimulating factor priming chemotherapy is more effective than standard chemotherapy as salvage therapy in relapsed acute myeloid leukemia.

    Science.gov (United States)

    Shen, Ying; He, Aili; Wang, Fangxia; Bai, Ju; Wang, Jianli; Zhao, Wanhong; Zhang, Wanggang; Cao, Xingmei; Chen, Yinxia; Liu, Jie; Ma, Xiaorong; Chen, Hongli; Feng, Yuandong; Yang, Yun

    2017-12-29

    To improve the complete remission (CR) rate of newly diagnosed acute myeloid leukemia (AML) patients and alleviate the severe side effects of double induction chemotherapy, we combined a standard regimen with granulocyte colony-stimulating factor (G-CSF) priming chemotherapy to compose a new double induction regimen for AML patients who failed to achieve CR after the first course. Ninety-seven patients with AML who did not achieve CR after the first course of standard chemotherapy were enrolled. Among them, 45 patients received G-CSF priming combined with low-dose chemotherapy during days 20-22 of the first course of chemotherapy, serving as priming group, 52 patients were administered standard chemotherapy again, serving as control group. Between the two groups there were no differences in the French-American-British (FAB) classification, risk status, the first course of chemotherapy, blood cell count or blasts percentage of bone marrow before the second course. But the CR rate was significantly higher and the adverse effect was much lower in the priming group than the control group. Cox multivariate regression analysis showed that WBC level before the second course and the selection of the second chemotherapy regimen were two independent factors for long survival of patients. These results elucidate that standard chemotherapy followed by G-CSF priming new double induction chemotherapy is an effective method for AML patients to improve CR rate and reduce adverse effects. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  1. Both IL-1β and TNF-α Regulate NGAL Expression in Polymorphonuclear Granulocytes of Chronic Hemodialysis Patients

    Directory of Open Access Journals (Sweden)

    Adriana Arena

    2010-01-01

    Full Text Available Background. NGAL is involved in modulation of the inflammatory response and is found in the sera of uremic patients. We investigated whether hemodiafiltration (HDF could influence the ability of polymorphonuclear granulocytes (PMGs to release NGAL. The involvement of interleukin- (IL-1β and tumor necrosis factor- (TNF-α on NGAL release was evaluated. Methods. We studied end-stage renal disease (ESRD patients at the start of dialysis (Pre-HDF and at the end of treatment (Post-HDF and 18 healthy subjects (HSs. Peripheral venous blood was taken from HDF patients at the start of dialysis and at the end of treatment. Results. PMGs obtained from ESRD patients were hyporesponsive to LPS treatment, with respect to PMG from HS. IL-1β and TNF-α produced by PMG from post-HDF patients were higher than those obtained by PMG from pre-HDF. Neutralization of IL-1β, but not of TNF-α, determined a clear-cut production of NGAL in PMG from healthy donors. On the contrary, specific induction of NGAL in PMG from uremic patients was dependent on the presence in supernatants of IL-1β and TNF-α. Conclusion. Our data demonstrate that in PMG from healthy subjects, NGAL production was supported solely by IL-1β, whereas in PMG from HDF patients, NGAL production was supported by IL-1β, TNF-α.

  2. Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Riklis, I; Kletter, Y; Bleiberg, I; Fabian, I

    1989-04-01

    The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

  3. Mobilization of stem cell with granulocyte-colony stimulating factor promotes recovery after traumatic brain injury in rat

    Directory of Open Access Journals (Sweden)

    Mohsen Marzban

    2010-01-01

    Full Text Available Introduction: This study was designed to investigate the effects of granulocyte colony-stimulating factor (G-CSF administration in rats for 6 weeks after traumatic brain injury (TBI. Methods: Adult male Wistar rats (n = 30 were injured with controlled cortical impact device and divided into four groups. The treatment groups (n = 10 each were injected subcutaneously with recombinant human G-CSF. Vehicle group (n=10 received phosphate buffered saline (PBS and only Brdu intraperitoneally. Bromodeoxyuridine (BrdU was used for mitotic labeling. Experimental rats were injected intraperitoneally with BrdU. Rats were killed at 6th week after traumatic brain injury. Neurological functional evaluation of animals was performed before and after injury using neurological severity scores (NSS. Animals were sacrificed 42 days after TBI and brain sections were stained using Brdu immunohistochemistry. Results: Statistically significant improvement in functional outcome was observed in treatment groups when compared with control (p<0.01. This benefit was visible 7 days after TBI and persisted until 42 days (end of trial. Histological analysis showed that Brdu cell positive was more in the lesion boundary zone at treatment animal group than all injected animals. Discussion: We believe that G-CSF therapeutic protocol reported here represents an attractive strategy for the development of a clinically significant noninvasive traumatic brain injury therapy.

  4. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    International Nuclear Information System (INIS)

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F.

    1990-01-01

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125 I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB- 125 I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

  5. Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

    International Nuclear Information System (INIS)

    Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

    1988-01-01

    The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

  6. The role of granulocyte macrophage colony stimulating factor (GM-CSF) in radiation-induced tumor cell migration.

    Science.gov (United States)

    Vilalta, Marta; Brune, Jourdan; Rafat, Marjan; Soto, Luis; Graves, Edward E

    2018-03-13

    Recently it has been observed in preclinical models that that radiation enhances the recruitment of circulating tumor cells to primary tumors, and results in tumor regrowth after treatment. This process may have implications for clinical radiotherapy, which improves control of a number of tumor types but which, despite continued dose escalation and aggressive fractionation, is unable to fully prevent local recurrences. By irradiating a single tumor within an animal bearing multiple lesions, we observed an increase in tumor cell migration to irradiated and unirradiated sites, suggesting a systemic component to this process. Previous work has identified the cytokine GM-CSF, produced by tumor cells following irradiation, as a key effector of this process. We evaluated the ability of systemic injections of a PEGylated form of GM-CSF to stimulate tumor cell migration. While increases in invasion and migration were observed for tumor cells in a transwell assay, we found that daily injections of PEG-GM-CSF to tumor-bearing animals did not increase migration of cells to tumors, despite the anticipated changes in circulating levels of granulocytes and monocytes produced by this treatment. Combination of PEG-GM-CSF treatment with radiation also did not increase tumor cell migration. These findings suggest that clinical use of GM-CSF to treat neutropenia in cancer patients will not have negative effects on the aggressiveness of residual cancer cells. However, further work is needed to characterize the mechanism by which GM-CSF facilitates systemic recruitment of trafficking tumor cells to tumors.

  7. Peripheral Blood CD64 Levels Decrease in Crohn’s Disease following Granulocyte and Monocyte Adsorptive Apheresis

    Directory of Open Access Journals (Sweden)

    Toshimi Chiba

    2011-12-01

    Full Text Available Granulocyte and monocyte adsorptive apheresis (GMA is reportedly useful as induction therapy for Crohn’s disease (CD. However, the effects of GMA on CD64 have not been well characterized. We report here our assessment of CD64 expression on neutrophils before and after treatment with GMA in two patients with CD. The severity of CD was assessed with the CD activity index (CDAI. The duration of each GMA session was 60 min at a flow rate of 30 ml/min as per protocol. CD64 expression on neutrophils was measured by analyzing whole blood with a FACScan flow cytometer. In case 1, CD64 levels after each session of GMA tended to decrease compared to pretreatment levels, whereas in case 2, CD64 levels dropped significantly after treatment. The CDAI decreased after GMA in both cases 1 and 2. A significant correlation was noted between CDAI scores and CD64 levels in both cases. In conclusion, GMA reduced blood CD64 levels, which would be an important factor for the decrease of CDAI scores.

  8. Granulocyte colony-stimulating factor in toxic epidermal necrolysis (TEN) and Chelsea & Westminster TEN management protocol [corrected].

    Science.gov (United States)

    de Sica-Chapman, A; Williams, G; Soni, N; Bunker, C B

    2010-04-01

    Toxic epidermal necrolysis (TEN) is a rare but life-threatening, allergic drug reaction. Skin blistering with epidermal and mucosal necrolysis with subsequent detachment from an inflamed underlying dermis is a hallmark of the condition. The pathogenesis of TEN is not well understood, accounting for controversies about its management and significant delay in initiating potentially beneficial therapy. There are no management protocols based on a robust evidence base. Prompt recognition of the diagnosis and consensus on early management initiatives are necessary in order to improve outcomes and survival in TEN. To date, TEN management has been directed at arresting the allergic reaction and treating the complications. We have identified a need for specific medical interventions to accelerate wound regeneration. This approach has not previously been adopted in the management of TEN. We observed that in two cases of severe TEN, dramatic re-epithelialization and recovery coincided with the introduction of granulocyte colony-stimulating factor (G-CSF) for neutropenia. We explain how addition of the G-CSF promotes recovery from TEN by enhanced bioregeneration of the damaged tissues through accelerated re-epithelialization. G-CSF has been used for severe neutropenia in TEN, but we recommend and explain why, as in our Chelsea and Westminster protocol, G-CSF should be considered in treating severe TEN irrespective of the severity of neutropenia.

  9. Treatment of blastic transformation of chronic granulocytic leukemia by chemotherapy, total body irradiation and infusion of cryopreserved autologous marrow

    Energy Technology Data Exchange (ETDEWEB)

    Buckner, C D; Stewart, P; Clift, R A; Fefer, A; Neiman, P E; Singer, J; Storb, R; Thomas, E D [Washington Univ., Seattle (USA). School of Medicine; The United States Public Health Service Hospital; Providence Medical Center, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA)

    1978-01-01

    We have previously reported attempts to reestablish the chronic phase of chronic granulocytic leukemia (CGL), in two patients with blastic transrormation, utilizing intensive therapy followed by the infusion of cryopreserved autologous marrow. This approach has now been attempted in a total of seven patients. Marrow was harvested on single or multiple occasions during the chronic phase of CGL and cryopreserved in 10% dimethylsulfoxide. All patients were treated with cyclophosphamide, 120 mg/kg, plus 1,000 rad of total body irradiation followed by infusion of stored marrow. Two patients failed to achieve marrow repopulation and died of infection after 29 and 48 days. Three patients had partial marrow recovery. Two of these achieved repopulation of myeloid, erythroid, and lymphoid elements but did not recover platelet function; one died of hemorrhage on day 55, and one died of cytomegalovirus interstitial pneumonitis on day 58. A third patient had delayed engraftment of all cell elements, most prominently lymphocytes, and died after 84 days of an iodopathic interstitial pneumonitis. Two patients achieved prompt and complete reestablishment of the chronic phase of CGL. One died on day 72 with a fungal pheumonitis and one developed blastic transformation within 4 months. These preliminary results indicate that this approach to the treatment of blastic transformation of CGL is feasible but difficult. Improvements in results may be achieved by more frequent storage of marrow and pheripheral blood stem cells and lymphocytes and further advances in pretransplant therapy.

  10. Intranasal Delivery of Granulocyte Colony-Stimulating Factor Enhances Its Neuroprotective Effects Against Ischemic Brain Injury in Rats.

    Science.gov (United States)

    Sun, Bao-Liang; He, Mei-Qing; Han, Xiang-Yu; Sun, Jing-Yi; Yang, Ming-Feng; Yuan, Hui; Fan, Cun-Dong; Zhang, Shuai; Mao, Lei-Lei; Li, Da-Wei; Zhang, Zong-Yong; Zheng, Cheng-Bi; Yang, Xiao-Yi; Li, Yang V; Stetler, R Anne; Chen, Jun; Zhang, Feng

    2016-01-01

    Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor with strong neuroprotective properties. However, it has limited capacity to cross the blood-brain barrier and thus potentially limiting its protective capacity. Recent studies demonstrated that intranasal drug administration is a promising way in delivering neuroprotective agents to the central nervous system. The current study therefore aimed at determining whether intranasal administration of G-CSF increases its delivery to the brain and its neuroprotective effect against ischemic brain injury. Transient focal cerebral ischemia in rat was induced with middle cerebral artery occlusion. Our resulted showed that intranasal administration is 8-12 times more effective than subcutaneous injection in delivering G-CSF to cerebrospinal fluid and brain parenchyma. Intranasal delivery enhanced the protective effects of G-CSF against ischemic injury in rats, indicated by decreased infarct volume and increased recovery of neurological function. The neuroprotective mechanisms of G-CSF involved enhanced upregulation of HO-1 and reduced calcium overload following ischemia. Intranasal G-CSF application also promoted angiogenesis and neurogenesis following brain ischemia. Taken together, G-CSF is a legitimate neuroprotective agent and intranasal administration of G-CSF is more effective in delivery and neuroprotection and could be a practical approach in clinic.

  11. Two protocols to treat thin endometrium with granulocyte colony-stimulating factor during frozen embryo transfer cycles.

    Science.gov (United States)

    Xu, Bin; Zhang, Qiong; Hao, Jie; Xu, Dabao; Li, Yanping

    2015-04-01

    The efficacy of two granulocyte colony-stimulating factor (G-CSF) protocols for thin endometrium were investigated. Eighty-two patients were diagnosed with thin endometrium (endometrial scratch subgroups. Compared with previous cycles, endometrial thickness increased from 5.7 ± 0.7 mm to 8.1 ± 2.1 mm after G-CSF treatment (P Endometrial thickness increases were not significantly different between the two subgroups. The G-CSF with endometrial scratch subgroup established nominally higher though non-significant clinical pregnancy and live birth rates than the G-CSF only subgroup (53.8 % versus 42.9% and 38.5% versus 28.6%, respectively). Fifty-two patients underwent FET despite edometrial thickness less than 7 mm, and were included as controls. Significantly higher embryo implantation and clinical pregnancy rates were observed in the G-CSF group compared with the control group (31.5% versus 13.9%; P Endometrial scracth did not impair G-CSF treatment for thin endometrium and favoured pregnancy and live birth rates. For patients with thin endometrium, embryo transfer cancellation and G-CSF treatment in subsequent FET cycles is beneficial. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. Sarcoma granulocítico multicêntrico como recidiva de leucemia mieloide aguda Multicentric granulocytic sarcoma as relapse of acute myelogenous leukemia

    Directory of Open Access Journals (Sweden)

    Taciana G. S. Aguiar

    2009-01-01

    Full Text Available Sarcoma granulocítico (SG é um tumor sólido extramedular, constituído por células precursoras de granulócitos. É geralmente associado a leucemia mieloide aguda ou raramente a outras desordens mieloproliferativas. O tumor geralmente ocorre precedendo uma leucemia mieloide aguda, durante o seu curso ou após a remissão ter sido alcançada. O prognóstico é pobre e tem como principais modalidades terapêuticas a quimioterapia e a radioterapia. Relata- se um caso de SG multicêntrico, de evolução rápida, com acometimento difuso de pele, mamas, gânglios linfáticos, tecido celular subcutâneo e líquor, em mulher de 45 anos, fora de tratamento para leucemia mieloide aguda e em remissão hematológica há 18 meses. A paciente apresentava dor intensa em membro inferior direito há uma semana e estava em anticoagulação oral há seis meses por trombose venosa profunda neste membro. Diagnosticado o SG, a paciente foi tratada com radioterapia e quimioterapia com boa resposta. Após três meses de seguimento, em vigência do tratamento quimioterápico, evoluiu com recidiva do SG neste membro, associado ao acometimento das mamas e posteriormente do sistema nervoso central, evoluindo para óbito em aplasia e sepses.Granulocytic sarcoma is an extramedullary solid tumor consisting of immature granulocytic cells. It is often associated with acute myelogenous leukemia and more rarely with other myeloproliferative disorders. The tumor generally occurs before acute myeloid leukemia, during its course or after disease remission. It has a poor prognosis with the main therapeutic options being chemotherapy and radiotherapy. A multicentric accelerated case of granulocytic sarcoma of a 45- year- old woman with diffuse skin, breast, lymphatic ganglia and subcutaneous tissue presentations no longer undergoing treatment for acute myeloid leukemia and in hematologic remission for 18 months is reported. The patient presented with severe pain of right lower

  13. Predictive value of the flow cytometric PCNA - assay (proliferating cell nuclear antigen) in head and neck tumors after accelerated-hyperfractionated radiochemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wenz, F; Lohr, F; Rudat, V; Dietz, A; Flentje, M; Wannenmacher, M

    1995-07-01

    Purpose/Objective: Proliferation of surviving tumor cells during fractionated radiotherapy may limit tumor control, especially in rapidly proliferating tumors. It has been widely accepted, that this may play a major role in head and neck tumors. Several methods for the assessment of tumor proliferation have been developed, however, most of them are either laborious, invasive or potentially toxic. Today, the gold standard is the flow cytometric BrdUrd assay. We present a flow cytometric method for detection of PCNA, which is an intranuclear proliferation associated protein, in solid human head and neck tumors and how these data correlate with outcome. Materials and Methods: Pretherapeutic biopsies of 20 inoperable patients with squamous cell carcinoma of the head and neck (T3-4N2M0) were examined. The tissue was disaggregated with pepsin/HCl, antibody staining was performed using the clone PC10. Biparametric flow cytometry was performed after a FITC conjugated secondary antibody and propidiumjodine staining was applied. The PCNA-index (i.e. percentage PCNA-positive cells), the DNA-index and the S-phase fraction (SPF, euploid tumors only) were determined. The therapy consisted of combined accelerated-hyperfractionated radiochemotherapy (66 Gy in 5 wks, concomittant boost of 1.6 Gy/d in wks 4+5, Carboplatin in wks 1+5). The median follow-up time was 14 mths (5 - 28), the clinical partners (V.R., A.D.) were 'blinded' towards the PCNA-values. Results: 13 patients suffered from disease progession and 11 died. The actuarial median survival and disease free survival (DFS) were 14.4 and 10.7 mths, respectively. The PCNA-values ranged from 3.2 to 70% (median 9%), there were 7 aneuploid and 13 euploid tumors. SFP in the euploid tumors ranged from 4 to 14.5% (median 10.5%). Neither SFP nor ploidy had a significant influence on the outcome. The patients were divided according to their PCNA-value in higher (n=10) and lower (n=10) than the median. The survival and DFS were 13

  14. Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

    Science.gov (United States)

    Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

    2011-07-18

    Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

  15. [Standardization of the quantitative flow cytometric test with anti-D antibodies for fetomaternal hemorrhage in RhD negative women].

    Science.gov (United States)

    Spychalska, Justyna; Uhrynowska, Małgorzata; Pyl, Hanna; Klimczak-Jajor, Edyta; Kopeć, Izabella; Peciakowska, Małgorzata; Gutowska, Renata; Gawlak, Maciej; Słomska, Sylwia; Dąbkowska, Syiwia; Szczecina, Roman; Dębska, Marzena; Brojer, Ewa

    2015-07-01

    In order to determine the appropriate dose of anti-D immunoglobulin to be administered as a preventive measure against hemolytic disease of the fetus/newborn in the subsequent pregnancy it is necessary to assess the number of fetal red blood cells that infiltrate/penetrate into the maternal circulation as a result of fetomaternal hemorrhage (FMH). One of the quantitative methods of FMH analysis is based on flow cytometry (FACS) which makes use of monoclonal antibodies to RhD antigen (anti-D test). The aim of the study was to further develop the method, evaluate its sensitivity and reproducibility and to compare it with the test based on the detection of fetal hemoglobin (HbF). The FACS study involved 20 RhD negative pregnant women and 80 RhD negative women after delivery. The following monoclonal antibodies were used: BRAD 3 FITC (anti-RhD antigen), CD45 PerCP (anti leukocyte antigen CD45), and anti-HbF PE. The fluorescence intensity of cells incubated with BRAD 3 FITC was demonstrated to depend on the RhD antigen expression, though the anti-D test also detects the weak D variant. The CD45 PerCP antibodies increased the sensitivity of anti-D test since they eliminated the leukocytes which non-specifically bind anti-D from the analysis. The presence of anti-D antibodies in maternal plasma does not affect the quantitative assessment of the fetal RhD positive fetal cells with BRAD 3 FITC. In case of FMH, the results of the anti-D test were similar to those with anti-HbF antibodies. The flow cytometric test with anti-D and anti-CD45 is useful in the assessment of the fetomaternal hemorrhage in RhD negative women. The sensitivity of the test is estimated at 0.05%.

  16. Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis.

    Science.gov (United States)

    Zhou, Fangbin; Zhou, Yaying; Yang, Ming; Wen, Jinli; Dong, Jun; Tan, Wenyong

    2018-01-01

    Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reli