scholarly journals Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

2021 ◽  
Vol 49 (6) ◽  
pp. 412-418
Author(s):  
A. S. Sustretov ◽  
V. V. Bogush ◽  
O. S. Guseva ◽  
P. V. Iliasov ◽  
L. V. Limareva
Keyword(s):  
Flow Cytometry ◽  
Functional State ◽  
Human Blood ◽  
Immune Cells ◽  
Proliferative Activity ◽  
Interference Microscopy ◽  
Fluorescent Label ◽  
Dna Cytometry ◽  
Blood Lymphocytes ◽  
Flow Cytofluorimetry

Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10  healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200  Pro with a  specialized NanoQuant Plate™; 2)  by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a  modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a  decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by  55% vs baseline (409.8±22.3  ng/μL and 264.3±25.0  ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a  difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes. 

Fluorescent Methods in the Study of UV-Induced Changes in Structural and Functional State of Human Blood Lymphocytes

2012 ◽  
Vol 153 (6) ◽  
pp. 908-912
Author(s):  
V. G. Artyukhov ◽  
O. V. Putintseva ◽  
V. A. Vdovina ◽  
M. V. Pashkov ◽  
D. V. Vasilenko
Keyword(s):  
Functional State ◽  
Human Blood ◽  
Blood Lymphocytes ◽  
Human Blood Lymphocytes ◽  
Induced Changes

The Effect of Dannomycin on the Proliferative Activity of Stem-cells Developed from Human Lymphocytes

1964 ◽  
Vol 50 (6) ◽  
pp. 477-480 ◽  
Author(s):  
Graciela Costa ◽  
Giovanni Astaldi
Keyword(s):  
Stem Cells ◽  
Tissue Culture ◽  
Human Blood ◽  
Proliferative Activity ◽  
Human Lymphocytes ◽  
Somatic Cells ◽  
Inhibitory Effect ◽  
Blood Lymphocytes ◽  
Human Blood Lymphocytes ◽  
Small Doses

Dannomycin is an antibiotic which interferes with the proliferative activity of somatic cells. The effect of Dannomycin on the proliferative activity of stem-cells developed from human blood lymphocytes in tissue culture with phytohemagglutinin was studied. The degree of proliferation was evaluated by the stathmokinetic test. Daunomycin caused an inhibitory effect, but the degree of inhibition was not proportional to the dose of the drug. Small doses, such as 1 jig/ml reduced the degree of proliferation by one third, whereas 3 fig/ml reduced that activity to one third in respect to the control.

scholarly journals POS0369 ELEVATED EXPRESSION OF TIM-3 ON NEUTROPHILS CORRELATES WITH DISEASE ACTIVITY AND SEVERITY OF ANKYLOSING SPONDYLITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 414.2-415
Author(s):  
X. Huang ◽  
T. W. Li ◽  
J. Chen ◽  
Z. Huang ◽  
S. Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ankylosing Spondylitis ◽  
Disease Activity ◽  
Immune Cells ◽  
Clinical Manifestations ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Bacterial Killing ◽  
Markers Of Inflammation ◽  
Significant Difference

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared

scholarly journals Mouse IgG3 binding to macrophage-like cells is prevented by deglycosylation of the antibody or by Accutase treatment of the cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alicja Karabasz ◽  
Monika Bzowska ◽  
Joanna Bereta ◽  
Maria Czarnek ◽  
Maja Sochalska ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Complex Network ◽  
Immune Cells ◽  
Specific Receptor ◽  
Fcγ Receptors ◽  
Specific Receptors ◽  
First Time

AbstractThe binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

Study of interaction between l-glutamate and human blood lymphocytes

1997 ◽  
Vol 58 (3) ◽  
pp. 177-180 ◽  
Author(s):  
I.A Kostanyan ◽  
M.I Merkulova ◽  
E.V Navolotskaya ◽  
R.I Nurieva
Keyword(s):  
Human Blood ◽  
Blood Lymphocytes ◽  
Human Blood Lymphocytes

A simplified and reliable assay for complex I in human blood lymphocytes

2007 ◽  
Vol 326 (1-2) ◽  
pp. 76-82 ◽  
Author(s):  
L.E.A. de Wit ◽  
L. Spruijt ◽  
G.C. Schoonderwoerd ◽  
I.F.M. de Coo ◽  
H.J.M. Smeets ◽  
...  
Keyword(s):  
Human Blood ◽  
Complex I ◽  
Blood Lymphocytes ◽  
Human Blood Lymphocytes

scholarly journals Carbon Nanotubes—Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 453
Author(s):  
Camilla Kofoed Andersen ◽  
Sangita Khatri ◽  
Jonas Hansen ◽  
Sofie Slott ◽  
Rohith Pavan Parvathaneni ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Carbon Nanotubes ◽  
B Cells ◽  
Human Blood ◽  
Immune Cells ◽  
Bone Marrow Cells ◽  
Coupling Efficiency ◽  
Drug Carriers ◽  
Whole Body ◽  
Whole Body Imaging

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77–79% for HiPco-SWCNT and 71–83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90–97% for HiPco-SWCNT and 87–98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000–1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

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