scholarly journals Formation of extracellular traps by circulating neutrophils and monocytes in rheumatoid arthritis patients: a study of new citrullinated autoantigen

2021 ◽  
Vol 23 (5) ◽  
pp. 1165-1170
Author(s):  
S. A. Bedina ◽  
E. E. Mozgovaya ◽  
A. S. Trofimenko ◽  
S. S. Spitsyna ◽  
M. A. Mamus
Keyword(s):  
Gradient Centrifugation ◽  
Autoimmune Response ◽  
Healthy Controls ◽  
Trypan Blue Exclusion ◽  
Autoantibody Production ◽  
Extracellular Traps ◽  
Trypan Blue Exclusion Test ◽  
Blood Neutrophils ◽  
Morphological Differences

Detection of subcellular structures containing typical citrullinated rheumatoid autoantigens in a single compartment presents a special interest, due to importance of anticitrulline autoantibodies for the autoimmune response in RA. Neutrophil and monocyte extracellular traps (NETs and ETs, respectively) may be considered such candidate structures. Our objective was to assess ability of blood neutrophils and monocytes from RA patients to generate NETs and ETs spontaneously and after in vitro induction.32 patients with verified RA and 30 healthy volunteers as controls were included into the study. Circulating neutrophils and monocytes were isolated with one-step density gradient centrifugation using three layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated microscopically using Trypan Blue exclusion test, as well as Nitro-Blue Tetrazolium test. The NETs were induced by phorbol-12-myristate-13-acetate, and ETs by bacterial LPS. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy. Neutrophil and monocyte fractions contained minute percentages of impurities and low extents of activated and dead cells. Spontaneous NET and ET formation in RA patients was significantly increased comparing to healthy controls. Neutrophils from ACPA-positive RA patients were found to have higher frequency of NET formation, compared to ACPA-negative RA patients. The monocytes did not demonstrate such differences between these subgroups. There were no substantial morphological differences in NETs and ETs patterns between the individuals from both groups. Induced extracellular trap production in RA was significantly higher compared to healthy controls. The level of myeloperoxidase-specific fluorescence in ETs was considerably lower than in NETs. NETs could probably be considered as a source of citrulline autoantigen participating in autoantibody production and stimulation of inflammatory autoimmune responses in RA, whereas ETs may play less important role in this process.

scholarly journals Suitability of a Progenitor Cell-Enriching Device for In Vitro Applications

Coatings ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 146
Author(s):  
Antonio Celentano ◽  
Tami Yap ◽  
Giuseppe Pantaleo ◽  
Rita Paolini ◽  
Michael McCullough ◽  
...  
Keyword(s):  
Cell Viability ◽  
Time Course ◽  
Ex Vivo ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Initial Reduction ◽  
Metal Wear ◽  
Class 1 ◽  
Electron Energy Loss

Rigenera® is a novel class-1 medical device that produces micro-grafts enriched of progenitors cells without ex vivo manipulation of donor tissues. The manufacturer’s protocol has been supported for a wide variety of clinical uses in the field of regenerative medicine. This study aimed to evaluate its potential use for in vitro cell models. Human primary oral fibroblasts were cultured under standard conditions and processed through Rigenera® over a time course of up to 5 min. Cell viability was assessed using a Trypan Blue exclusion test. It is possible to process fibroblasts through Rigenera® although an initial reduction of cell viability was observed. Additionally, debris was evident in the cell suspension of the processed samples. Scanning electron microscopy (SEM) microanalysis of the debris and electron energy-loss spectroscopy confirmed the presence of metal wear possibly due to the processing conditions used in this study. Interestingly, pore sizes within Rigeneracons® grids were found to range between 250–400 μm. This is the first report assessing the suitability of Rigenera® and Rigeneracons® for in vitro applications. Whilst Rigenera® workflow was found to be amenable to laboratory uses, our results strongly suggest that further research and development is necessary to support the utilization of this technology for enrichment of micro-graft derived cells and cell sorting in vitro.

scholarly journals In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay

2006 ◽  
Vol 17 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Daniel Araki Ribeiro ◽  
Mariângela Esther Alencar Marques ◽  
Daisy Maria Fávero Salvador
Keyword(s):  
Comet Assay ◽  
Single Cell ◽  
Mammalian Cells ◽  
Lymphoma Cells ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Gutta Percha ◽  
Mouse Lymphoma Cells ◽  
Mouse Lymphoma

Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 muL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

In vitro photoradiation therapy of the rat 9L gliosarcoma

1986 ◽  
Vol 64 (5) ◽  
pp. 775-779 ◽  
Author(s):  
Douglas Hamilton ◽  
John D. S. McKean ◽  
John Tulip ◽  
Donald Boisvert ◽  
Judy Cummins
Keyword(s):  
Trypan Blue ◽  
Glioma Cells ◽  
Laser Source ◽  
Trypan Blue Exclusion ◽  
Content Type ◽  
9L Gliosarcoma ◽  
Trypan Blue Exclusion Test ◽  
9L Glioma ◽  
Fine Print

✓ The authors have investigated various factors involved in the photoradiation treatment of 9L glioma cells. The cells were grown in tissue culture and exposed to light from a laser source that allowed accurate quantitation of the light energy. Cell death was determined following treatment using the trypan blue exclusion test. It was shown that the treatment is very wavelength-dependent following the absorption spectrum of hematoporphyrin derivative (HPD). The absorption peaks in the lower part of the spectrum are more efficient than those of higher wavelengths. Photoradiation therapy is more effective the higher the concentration of HPD. Intensity of light is a very important factor in calculating the total dose of light necessary for this treatment.

Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus

1992 ◽  
Vol 66 (2) ◽  
pp. 124-131 ◽  
Author(s):  
D. Janssen ◽  
A. Osuna ◽  
J. Lazuen ◽  
P. H. De Rycke
Keyword(s):  
Hydatid Cyst ◽  
Echinococcus Granulosus ◽  
Peritoneal Macrophages ◽  
Toxic Substances ◽  
Hydatid Cysts ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Concomitant Immunity ◽  
Immunological Reactions

ABSTRACTInfection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.

Polygalae Radix Extract Protects Cultured Rat Granule Cells Against Damage Induced by NMDA

2004 ◽  
Vol 32 (04) ◽  
pp. 599-610 ◽  
Author(s):  
Hyun Joo Lee ◽  
Ju Yeon Ban ◽  
Sang Bum Koh ◽  
Nak Sul Seong ◽  
Kyung Sik Song ◽  
...  
Keyword(s):  
Cell Death ◽  
Granule Cells ◽  
Cell Damage ◽  
Glutamate Release ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Inhibitory Effect ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test

Polygalae Radix (PR) from Polygala tenuifolia (Polygalaceae) is traditionally used in China and Korea, as this herb has a sedative, anti-inflammatory and antibacterial agent. To extend our understanding of the pharmacological actions of PR in the CNS on the basis of its CNS inhibitory effect, the present study examined whether PR has the neuroprotective action against N-methyl-D-aspartate (NMDA)-induced cell death in primarily cultured rat cerebellar granule neurons. PR, over a concentration range of 0.05 to 5 μg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. PR (0.5 μg/ml) inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pre-treatment of PR (0.5 μg/ml) inhibited NMDA (1 mM)-induced elevation of intracellular Ca 2+ concentration ([ Ca 2+] i ), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that PR prevents NMDA-induced neuronal cell damage in vitro.

CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3428-3430 ◽  
Author(s):  
Olivier Boyer ◽  
David Saadoun ◽  
Julien Abriol ◽  
Mélanie Dodille ◽  
Jean-Charles Piette ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C ◽  
Treg Cell ◽  
Mixed Cryoglobulinemia ◽  
Treg Cells ◽  
Healthy Controls ◽  
Cell Frequency ◽  
Autoantibody Production

Abstract Patients who are chronically infected with hepatitis C virus (HCV) often develop mixed cryoglobulinemia (MC), a B-cell proliferative disorder with polyclonal activation and autoantibody production. We investigated if MC is associated with a deficit of CD4+CD25+ immunoregulatory T (Treg) cells, which have been shown to control autoimmunity. Because Treg cells express higher amounts of CD25 than activated CD4+ T cells, we analyzed blood CD4+CD25high Treg cells in 69 untreated patients chronically infected with HCV. Treg cell frequency in patients without MC (8.8% ± 2.3%) or with asymptomatic MC (7.4% ± 2.1%) was comparable to that of healthy controls (7.9% ± 1.3%). In contrast, it was significantly reduced in symptomatic MC patients (2.6% ± 1.2%, P &lt; .001) even when compared to a panel of untreated HCV- patients with different inflammatory disorders (6.2% ± 0.8%, P &lt; .0001). In symptomatic MC patients, the purified remaining CD4+CD25+ T cells retained suppressive activity in vitro. These results, together with experimental data showing that depletion of Treg cells induces autoimmunity, suggest a major role of Treg cell deficiency in HCV-MC vasculitis and this is the first report of a quantitative Treg cell deficiency in virus-associated autoimmunity. (Blood. 2004; 103:3428-3430)

scholarly journals Defensin-rich dense granules of human neutrophils

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 757-765 ◽  
Author(s):  
WG Rice ◽  
T Ganz ◽  
JM Jr Kinkade ◽  
ME Selsted ◽  
RI Lehrer ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Human Neutrophils ◽  
Total Acid ◽  
Density Fractions ◽  
Dense Granules ◽  
Blood Neutrophils

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

scholarly journals The Involvement of Neutrophil Extracellular Traps in Disease Activity Associated With IgA Vasculitis

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiu-Qi Chen ◽  
Li Tu ◽  
Jia-Sen Zou ◽  
Shi-Qun Zhu ◽  
Yan-Jun Zhao ◽  
...  
Keyword(s):  
Disease Activity ◽  
Drug Withdrawal ◽  
Neutrophil Extracellular Traps ◽  
Dnase I ◽  
Healthy Children ◽  
Healthy Controls ◽  
Iga Vasculitis ◽  
Extracellular Traps ◽  
Circulating Levels

ObjectivesThis aim of this study was to determine whether neutrophil extracellular traps (NETs) are involved in the pathogenesis of IgA vasculitis (IgAV) and investigate whether the circulating NETs levels are associated with disease activity in children.MethodsWe performed a case-control study and collected blood samples from 193 children with different stages of IgAV (61 were at the onset stage, 64 at the remission stage, 43 at the active stage, and 25 were undergoing drug withdrawal). A total of 192 healthy children were recruited as controls. Circulating cell free DNA (cf-DNA) was obtained from the plasma and quantified by using the Quant-iT PicoGreen DNA quantification kit. NETs-associated myeloperoxidase-DNA (MPO-DNA), citrullinated-histone H3 (cit-H3), neutrophil elastase (NE), and the deoxyribonuclease I (DNase I) concentrations were measured using enzyme-linked immunosorbent assays. The presence of NETs in the kidney and gastrointestinal tissues of onset and active IgAV patients was determined by multiple immunofluorescence staining in 15 IgAV nephritis patients and 9 IgAV patients without IgAV nephritis, respectively. NETs degradation potency of collected sera samples from IgAV patients were checked in vitro. Relationships between circulating levels of cf-DNA with MPO-DNA, NE, and DNase I and the patients were analyzed.ResultsCirculating levels of cf-DNA in onset and active IgAV patients were significantly higher than those in remission and drug withdrawal patients as well as healthy controls. The results were similar for MPO-DNA and NE. The levels of circulating cf-DNA correlated significantly with MPO-DNA, NE and DNase I. A significantly decreased degradation of NETs from the onset and active IgAV patients was observed, but was normal in healthy controls. Furthermore, presence of NETs was also confirmed in all renal and gastrointestinal tissues obtained from the onset and active IgAV patients but not control samples.ConclusionsOur data showed that NETs were released into the circulation of IgAV patients and are involved in the disease activity. The circulating levels of NETs maybe used to assess disease severity in children with IgAV.

Thrombin induces Contraction And increased 51-Cr Release from Cultured Human Endothelial Cells

1979 ◽  
Author(s):  
K.S. Galdal ◽  
S.A. Evensen
Keyword(s):  
Endothelial Cells ◽  
Calf Serum ◽  
Linear Increase ◽  
Foetal Calf Serum ◽  
Human Endothelial Cells ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Release Assay

Injury to human endothelial cells(EC) in primary culture was evaluated by a 51-Cr release assay, phase contrast microscopy and the trypan blue exclusion test. Normal integrity of EC, was maintained and 51-Cr release showed a slow linear increase during 24h incubation with either RPMI 1640 supplemented with 20% foetal calf serum, glutamine and antibiotics(SCM) or normal human serum(NHS). Thrombin in a concentration as low as 0.1 IU/ml induced obvious contraction of EC incubated in SCM, but the cells remained fixed to the bottom of the wells. Cell contraction was maximal after 15min and disappeared within 4h. 51-Cr release increased 2-3 fold within a few minutes and remained increased during an incubation period of 4h. The injurious effect of thrombin was inhibited in SCM containing hirudin(l.7 u/ml) or in NHS. ADP(10-5 M), endotoxin (0.1mg/ml) and 5 vasoactive agents adrenalin 5.5 10-5, noradrenalin 5.9 10-5 M, histamine 9.10-4 M, bradykinin 7.5 10-7 M and serotonin 10-5 M) did not cause cellular injury. Cultured human EC are injured by thrombin. The other tested agents do not appear to induce direct injury in vitro, but may interact with cellular elements in the blood to produce the endothelial injury previously observed in vivo.

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