scholarly journals Comparison of Polymorphonuclear Cells from Healthy Donors and Differentiated HL-60 Cells as Phagocytes in an Opsonophagocytic Assay Using Antigen-Coated Fluorescent Beads

2000 ◽  
Vol 7 (2) ◽  
pp. 314-317 ◽  
Author(s):  
Bruno Guy ◽  
Catherine Testart ◽  
Sophie Gimenez ◽  
Violette Sanchez ◽  
Philippe Lheritier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Streptococcus Pneumoniae ◽  
Polymorphonuclear Cells ◽  
Vaccine Candidates ◽  
Specific Antibodies ◽  
Fluorescent Beads ◽  
Healthy Donors ◽  
Latex Beads ◽  
The Mean

ABSTRACT Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three- to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.

scholarly journals Use of Highly Encapsulated Streptococcus pneumoniae Strains in a Flow-Cytometric Assay for Assessment of the Phagocytic Capacity of Serotype-Specific Antibodies

1998 ◽  
Vol 5 (5) ◽  
pp. 703-710 ◽  
Author(s):  
W. T. M. Jansen ◽  
J. Gootjes ◽  
M. Zelle ◽  
D. V. Madore ◽  
J. Verhoef ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Streptococcus Pneumoniae ◽  
Surface Proteins ◽  
Heat Inactivation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphonuclear Cells ◽  
Phagocytic Capacity ◽  
Specific Antibodies ◽  
Flow Cytometric ◽  
Positive Results

ABSTRACT A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 μl per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

Quality control in neutrophil granulocyte (PMN) concentrates by flow cytometry

2005 ◽  
Vol 43 (7) ◽  
Author(s):  
Uwe Schwanke ◽  
Laura Schrader ◽  
Rainer Moog
Keyword(s):  
Flow Cytometry ◽  
Quality Control ◽  
Oxidative Burst ◽  
Neutrophil Granulocyte ◽  
Polymorphonuclear Cells ◽  
Autologous Plasma ◽  
Function Tests ◽  
Healthy Donors ◽  
Migration Capacity

AbstractBackground: In peripheral blood, chemotaxis, phagocytosis, and oxidative burst of polymorphonuclear cells (PMNs) can be assessed by flow cytometry, whereas function tests, i.e., quality control in PMN concentrates designed for neutropenia therapy, are lacking.Methods: PMN concentrates (n=6) harvested from healthy donors who had been premedicated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone were stored undiluted (control, C; n=6) and diluted 1:4 (D; n=6) with autologous plasma for 72h. Commercial flow cytometry function tests were performed to quantify changes in chemotaxis, phagocytosis, and oxidative burst of PMNs over time.Results: Median levels of phagocytosis and oxidative burst levelled at 86% (82–94) and 98% (83–100) in C on the day of apheresis, respectively, but deteriorated to 15% (0–24) and 0% within 72h; in D these parameters remained close to 90%. Median levels of chemotaxis were comparable in C (69%, 65–74) and D (74%, 70–84) at baseline. No migration was detected in C after 72h; however, D retained approximately 63% (13–76) migration capacity.Conclusion: Quality control in PMN concentrates is practical using flow cytometry and commercial test kits. While phagocytosis and oxidative burst may be maintained for 72h in vitro, chemotaxis of apheresed PMNs is already reduced on the day of apheresis.

scholarly journals Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Masahide Yano ◽  
Shruti Gohil ◽  
J. Robert Coleman ◽  
Catherine Manix ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Direct Effect ◽  
Pneumococcal Disease ◽  
Effector Cell ◽  
Capsular Polysaccharide ◽  
Genetic Exchange ◽  
Content Type ◽  
Specific Antibodies

ABSTRACTThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two differentStreptococcus pneumoniaeserotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotypeinvitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.IMPORTANCECurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology ofStreptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA

1987 ◽  
Author(s):  
J Grøndahl-HANSEN ◽  
N Agerlin ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mean Values ◽  
Amino Terminal ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Urokinase Type Plasminogen Activator ◽  
The Mean

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count &lt;20 copies/μL and 17.7% had a KREC count &lt;20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

scholarly journals Clinical Presentation and Bacterial Etiology of Adult Community Acquired Pneumonia

2017 ◽  
Vol 34 (3) ◽  
pp. 128-134
Author(s):  
Md Abdus Salam ◽  
Md Robed Amin ◽  
Quazi Tarikul Islam
Keyword(s):  
Streptococcus Pneumoniae ◽  
Blood Culture ◽  
Mycoplasma Pneumoniae ◽  
Chlamydia Pneumoniae ◽  
Community Acquired Pneumonia ◽  
Sputum Culture ◽  
Gram Positive ◽  
Gram Negative ◽  
The Mean ◽  
Gram Positive Cocci

Introduction: Pneumonia is a worldwide, serious threat to health and an enormous socio-economic burden for health care system. According to recent WHO data, each year 3-4 million patients die from pneumonia. The clinical presentations and bacterial agents responsible for community acquired pneumonia (CAP) varies according to geography and culture.Methods: A cross sectional observational study conducted among the 53 consecutive patients with a clinical diagnosis of CAP in admitted patient in the department of Medicine, DMCH, during January 2010 to December 2010. Hematological measurements (TC of WBC, Hb%, ESR, platelet count), blood culture, chest X-ray P/A view, sputum for Gram staining and culture sensitivity, sputum for AFB, blood urea and random blood sugar were done in all cases. ELISA for IgM antibody of Mycoplasma pneumoniae and Chlamydia pneumoniae were done in sputum culture negative cases.Results: The mean (±SD) age was 38.9±17.3 years and Male female ratio was 3:1. Fever, chest pain and productive cough were the most common clinical features. The mean (±SD) respiratory rate was 23.0±2.8 /minute . COPD and DM were found in 17.0% and 5.7% of patients respectively . Blood culture was found positive in only 1.9% of the study patients. Gram positive Cocci 62.26%, Gram negative Bacilli 9.43%, mixed Gram positive cocci and Gram negative bacilli 11.32% and Gram negative Cocco Bacilli 1.9% were observed and in 15.03 % cases, no bacteria could be seen. Sputum culture revealed 53.8% streptococcus pneumoniae, 26.9% Klebsiella pneumonia as predominant organism. Mycoplasma pneumoniae and Chlamydia pneumoniae were found in 7.4% and 3.7% respectively by serological test. For Streptococcus pneumoniae, sensitive antibiotics were Amoxyclav and Levofloxacin. For Gram negative bacilli and coccobacilli, more sensitive antibiotics were Meropenem, Ceftriaxone, and Clarithromycin. The best sensitive drug were found meropenem. The mean (±SD) duration of hospital stay was 5.0±1.7 days with ranging from 3 to 10 days.Conclusion: Region based bacteroiological diagnosis of Cap is important for selecting the best and sensitive drugs for complete cure.J Bangladesh Coll Phys Surg 2016; 34(3): 128-134

scholarly journals FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

2020 ◽  
Author(s):  
Lydia Horndler ◽  
Pilar Delgado ◽  
Ivaylo Balabanov ◽  
Georgina Cornish ◽  
Miguel Angel Llamas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Correct Identification ◽  
Truncated Form ◽  
Serological Tests ◽  
S Protein ◽  
Human Antibodies ◽  
Degree Of Protection ◽  
Immunoglobulin Isotypes ◽  
Jurkat T Cell ◽  
The Mean

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.

scholarly journals Innovator Filgrastim versus Generic Filgrastim in Hematopoietic Stem Cell Transplantation Mobilization

2021 ◽  
Author(s):  
Sadik Husian ◽  
Preethi Jeyaraman ◽  
S. K. Gupta ◽  
Reeta Rai ◽  
Sangeeta Pathak ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Healthy Donors ◽  
The Mean ◽  
Mean Time ◽  
Autologous Hsct

Abstract Methods This is a retrospective study. G-CSF was administered in the dose of 10 μg/kg subcutaneous as a single dose for 4 days. On day 5, peripheral blood stem cell (PBSC) apheresis was performed using Haemonetics MCS plus or COBE Spectra apheresis machine through a double-lumen central venous catheter. Primary outcome parameters were the total number of CD34+ HSCs/kg of recipient weight mobilized in peripheral blood and the number of days required for neutrophil and platelets engraftment, respectively. Objective We compared the effectiveness and safety of innovator filgrastim versus generic filgrastim in patients who underwent hematopoietic stem cell transplantation (HSCT). Results A total of 91 stem cell mobilizations was analyzed. There were 58 normal healthy donors for allogeneic HSCT and 33 patients for autologous HSCT. There was no statistically significant difference among groups in terms of total collected CD34+ cells value (p = 0.609). The mean time to neutrophil engraftment was 13.7 days in the innovator group and 13.2 days in the Grafeel group (p = 0.518). The mean time to platelet engraftment was 16.2 days in the innovator group and 14.8 days in the generic group (p = 0.435). The patient who received generic filgrastim had more febrile episodes during the course of transplantation (p = 0.020). Conclusion Generic filgrastim was found to be comparable to original filgrastim for peripheral blood stem cell mobilization in normal healthy donors for allogeneic HSCT and patients for autologous HSCT.

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