scholarly journals Novel approaches to increase resistance to acute respiratory infections

2021 ◽  
Vol 25 (3) ◽  
pp. 181-195
Author(s):  
Svetlana V. Guryanova ◽  
Natalia A. Kudryashova ◽  
Anastasiya A. Kataeva ◽  
Bubusaira T. Orozbekova ◽  
Natalia V. Kolesnikova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Medical Care ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Respiratory Infections ◽  
Maximum Efficiency ◽  
Physical Culture ◽  
Acute Respiratory Infections ◽  
Nonspecific Resistance ◽  
Healthy Donors

Relevance . Respiratory infections are the most common in the world. In order to prevent epidemics, there is a need to improve the strategies for organizing medical care and develop new approaches in order to increase the nonspecific resistance, mobilize innate immunity. Objective . The aim of this study was to investigate the effect of glucosaminylmuramyldipeptide (GMDP) on the level of expression of markers of differentiation and activation of functionally significant subpopulations of dendritic cells in peripheral blood mononuclear cells of healthy donors,the second aim was to assess the effectiveness of GMDP in the prevention of acute respiratory infections in an unfavorable epidemiological period of the COVID-19 pandemic. Materials and Methods . An open comparative study included 309 apparently healthy participants, aged 19-22 years. At the first stage of the study, 42 participants (22 female and 20 male) took the drug Licopid 1 mg for 10 days according to the instructions, 1 tablet 3 times a day in order to prevent acute respiratory infections. Peripheral blood sampling was performed before taking the drug (day 0) and the next day after the last dose of the drug (day 12). Evaluation of the expression of markers of differentiation and activation of dendritic cell subpopulations HLA-DR, CD11c, CD123, CD80, CD83, CCR7, CD3, CD14, CD20 was assessed by flow cytometry. At the same time, mRNA was isolated from mononuclear cells of perfusion blood and, after reverse transcription, the level of gene expression was determined by RT PCR. At the next stage, the effectiveness of the prophylactic use of the drug Licopid in 267 students of the Institute of Physical Culture was assessed in order to prevent acute respiratory infections in an unfavorable epidemiological period; the observation period was 12 months. Results and Discussion . A study of the relative quantitative composition of DCs in the peripheral blood of healthy donors by flow cytometry revealed the possibility of an increase in their total number, as well as subpopulations of MDC and PDC under the influence of GMDP. There was a statistically significant increase in the receptors for the chemokine CCR7, which is responsible for the recruitment of DCs to the secondary lymphoid organs. Analysis of the levels of expression of genes XCR1, CD11b , and CD103 showed a statistically significant effect of GMDP on an increase in their expression compared to the baseline level (before GMDP intake), with the mean value being higher in participants undergoing moderate exercise. It was found that the use of the drug Licopid 1mg for the purpose of preventing and reducing the seasonal incidence of acute respiratory infections at the stage of basic training of students of the Institute of Physical Culture contributed to a decrease in the incidence of acute respiratory infections within 12 months of observation after taking the drug. The number of episodes of acute respiratory infections decreased 3.7 times, while the group with 3 or more episodes of acute respiratory infections during the year, which constituted 14.5 % of participants, completely disappeared. The maximum efficiency of GMDP was observed in the track and field command, in which the number of participants who had no episodes of acute respiratory infections during the year increased by 7 times. Conclusion . Our data complement the modern understanding of the molecular mechanism of action of GMDP and substantiate the possibility of its experimental and clinical use in order to develop new strategies for organizing medical care in order to increase the nonspecific resistance of the organism.

scholarly journals Diagnostic criteria of lymphoproliferative diseases from the peripheral blood samples using a cell biochip

2021 ◽  
Vol 49 ◽  
Author(s):  
O. S. Fedyanina ◽  
Yu. Yu. Chuksina ◽  
A. N. Khmelevskaya ◽  
A. N. Khvastunova ◽  
Yu. N. Matveev ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Lymphoproliferative Disorders ◽  
Lower Proportion ◽  
Healthy Controls ◽  
Lymphoproliferative Diseases ◽  
Healthy Donors ◽  
A Cell

Background: At present, the diagnosis of lymphoproliferative disorders is based on the combination of blood or bone marrow smear morphology and immunophenotyping by flow cytometry. Immunophenotypic testing by flow cytometry technique is available only in big medical centers, which is not always convenient for a  patient. Therefore, development of an available method for preliminary diagnosis of lymphoproliferative diseases not requiring special equipment seems relevant.Materials and methods: Peripheral blood mononuclear cells from 17  patients admitted to the hospital with suspicion of a  lymphoproliferative disorder, and 17  healthy donors were studied on a cell biochip for determination of proportions of cells positive for various surface CD antigens. The diagnosis was verified by flow cytometry.Results: Compared to healthy controls and patients with T-cell lymphoproliferative disorders (TCLPD), the patients with B-cell lymphoproliferative disorders (BCLPD) had significantly lower proportion of CD7+ cells (medians, 7% and 73% respectively, p=2×10-6 for comparison with healthy controls; median  7% and 93% for comparison with TCLPD, p=0.032). In addition, the patients with BCLPD had higher proportion of peripheral СD19+ mononuclear cells, compared to that in the patients with TCLPD and healthy donors (medians 84% and 13% for comparison between BCLPD and healthy control, p=2×10-5; 84% and 3% for comparison of BCLPD and TCLPD, p=0.033). The patients with B-cell chronic lymphocytic leukemia had significantly higher CD5+ cells in the cell biochip compared to the patients with other BCLPD (medians 72% and 9%, p=0.024). The patients with TCLPD had significantly lower proportion of CD19+ cells than the healthy controls (medians, 3% and 13%, respectively, р=0.042).Conclusion: The study has demonstrated the potential to use the previously developed cell biochip for diagnosis of lymphoproliferative diseases. The biochip makes it possible to sort out white blood cells according to their surface differentiation antigen for their further morphological analysis. The cell biochip allows for the differential diagnosis between BCLPD and TCLPD and determination the lymphocyte clones based on the expression of immunoglobulin light chains.

scholarly journals Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

2021 ◽  
Author(s):  
Bo Li ◽  
Chunmei Yang ◽  
Gui Ja ◽  
Yansheng Liu ◽  
Na Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Effective Utilization ◽  
Blood Mononuclear Cells ◽  
Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

scholarly journals A two-color flow cytometry assay for detection of hairy cells using monoclonal antibodies

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1063-1068
Author(s):  
JS Kristensen ◽  
J Ellegaard ◽  
P Hokland
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Suspensions ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Morphological Evidence ◽  
Acute Leukemias ◽  
Color Flow ◽  
Hairy Cells

We have developed a simple two-color immunofluorescence assay equally suited for microscopy and flow cytometry detecting hairy cells (HCs) in single cell suspensions, based on the concomitant reactivities with the B cell-specific monoclonal antibody B1 (CD20) and the monocyte/HC- associated antibody SHCL-3 (CD11c). Thus, HCs can be demonstrated in peripheral blood, bone marrow, and spleen specimens from hairy cell leukemia (HCL) patients even when they constitute less than 1% of the cell suspension. Likewise, admixture experiments with normal mononuclear cells and the MOLT-4 T-acute lymphocytic leukemia (ALL) cell line demonstrated that HCs could be detected in amounts as low as 1%. The validity of this assay has been ascertained by the lack of double marker positivity in cell suspensions from B-chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) patients that only expressed B1 or SHCL-3, respectively. Furthermore, other malignant blood diseases, including malignant lymphomas, acute leukemias, and chronic leukemias disclosed no double marker positive cells. In a clinical setting, this assay was used for purifying HCs (by flow cytometry) from the peripheral blood from patients with no apparent morphological evidence of circulating HC infiltration and for monitoring the effect of interferon therapy. In conclusion, this assay should be of value for both diagnosis and monitoring patients with HCL.

scholarly journals Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry

PLoS ONE ◽  
2017 ◽  
Vol 12 (11) ◽  
pp. e0187440 ◽  
Author(s):  
Bo Langhoff Hønge ◽  
Mikkel Steen Petersen ◽  
Rikke Olesen ◽  
Bjarne Kuno Møller ◽  
Christian Erikstrup
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

scholarly journals Zika virus infects human blood mononuclear cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Carolina V. Messias ◽  
Julia P. Lemos ◽  
Daniela P. Cunha ◽  
Zilton Vasconcelos ◽  
Lidiane M. S. Raphael ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nervous System ◽  
Zika Virus ◽  
Mononuclear Cells ◽  
Health Concern ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Blood Mononuclear Cells ◽  
The Central Nervous System

Abstract Background Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. Methods PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. Results We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3−CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. Conclusions These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Discriminative HMGA2 Isoform Expression in CD15+ Granulocytic Cells in Myeloid Metaplasia with Myelofibrosis (MMM).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2429-2429
Author(s):  
Olivier Pierre-Louis ◽  
Joris Andrieux ◽  
Christophe Desterke ◽  
Eric Lippert ◽  
Vincent Praloran ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Full Length ◽  
Expression Level ◽  
Granulocytic Differentiation ◽  
Myeloid Metaplasia ◽  
Expression Levels ◽  
Healthy Donors ◽  
Exon 1 ◽  
Isoform Expression

Abstract MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34+ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated. RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15+ granulocytic cells (PBCD15+) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34+ cells (PBCD34+). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5). In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34+ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34+ MMM patients (p<10−6) compared to healthy donors. In MMM, HMGA2 expression level was significantly increased (p<10−5) in PBCD15+ as compared to PBCD34+. Moreover, PBCD15+ HMGA2 1–3 expression level was significantly higher in MMM patients compared to PBCD15+ from healthy donors (p<10−7). A persistence of HMGA2 1–5 expression was only observed in MMM PBCD15+ but was undetectable neither in normal PB neutrophils (purity>98%) nor in PB neutrophils from other myeloproliferative disorders (Polycythemia Vera and Essential Thrombocythemia). To determine if HMGA2 level was modified during hematopoietic differentiation, we quantified HMGA2 1–3 and 1–5 isoform expression on purified healthy donor PB CD34+ and MMM CD34+ before and after culture with specific lineage growth factors (12 day-culture). Primary results showed that both HMGA2 isoform expression levels were higher during granulocytic differentiation. Our results demonstrate that HMGA2 1–5 isoform is discriminately overexpressed in MMM PBCD15+. The persistence of this HMGA2 full length expression in MMM myeloid lineage could be considered as a marker of the disease.

Quantitative and Qualitative Analysis Of Regulatory T Cells (Tregs) Derived From The Peripheral Blood Of Chronic Lymphocytic Leukemia Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5280-5280
Author(s):  
Eleni Dikaia Ioannidou ◽  
Vassiliki Mpakou ◽  
Myrofora Vikentiou ◽  
Eugenia Konsta ◽  
Frieda Kontsioti ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Treg Cells ◽  
Lymphocytic Leukemia ◽  
Effector Cells ◽  
T Effector Cells ◽  
Healthy Donors

Abstract Introduction T regulatory cells are immunosuppressive cells, which are considered to play an important role in the regulation of immune response to cancer, by restraining autoreactive lymphocytes. Several studies, mostly in solid tumors, revealed that the number of Treg cells increases as the disease progresses and that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy, characterized by both, immunodeficiency and autoimmune disorders. Accumulated data indicate the role of T cells in the pathogenesis and development of CLL and reveal an increased number of Treg cells in CLL patients. The scope of this study is the analysis of the functional role of Tregs derived from the peripheral blood of CLL patients, mainly on B-CLL cells, and its correlation with well known prognostic factors. Methods Treg cells derived from mononuclear cells of 28 untreated B-cell CLL patients with a median age 62 (44-88) and 17 healthy donors were analyzed through Flow cytometry. Patients were classified according to Rai classification as Rai I:19, Rai II:4, Rai III:5 and according to Binet as Binet A: 24, Binet B:3 and Binet C:1. The following antibodies were used for the fluorescence-activated cell sorter (FACS) analysis: 1. CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7 2. CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7 3. CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7 4. beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7 5. Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7 Moreover, peripheral blood was obtained from 15 patients with B-cell CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+CD25+ (Treg cells), CD4+CD25- (T effectοr cells, Teff), CD5+CD19+ (B-CLL) and CD5+CD19- (Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL, NB cells, Tregs:Teff in a 4:1 ratio, B-cll:Tregs in 1:20 ratio, B-cll:Teff in 1:20 ratio, NB cells:Tregs in 1:20 ratio, NB cells:Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. Results FACS analysis of the Treg cells resulted at the following observations: (1) The co-expression of the CD45RA-CD45RO markers was significantly higher in patients’ samples than in controls (p=0.047). (2) No significant differences were observed between patients and controls, regarding the expression of the CD1α marker, as well as the expression of CD95 and CD152 markers. (3) The Treg absolute cell number (cells/μL), estimated either as the number of CD4+ CD25+ CD127- cells or as the number of CD4+ CD25+ FoxP3+ cells, was statistically significantly higher in patients’ samples than in controls (CD127- p=0.047, FoxP3+ p= 0.036). (4) Annexin V expression in Treg cells from B- CLL patients was significantly lower compared to controls (p=0.027). Following the purification and culturing of T and B cells from B-cell CLL patients’ samples, functional analysis of the different cell populations was performed using the BrdU proliferation assay. We observed that Tregs were able to significantly suppress the proliferation of the Teff cells (p=0.002). After the co-culturing of NB cells (CD5+CD19-)and Tregs (CD4+CD25+) we found that NB cells seemed to significantly increase the proliferation of Treg cells, compared to the proliferation capacity of the Tregs when cultured alone (p=0.047). Moreover, we observed that Teff (CD4+CD25-) were able to significantly suppress the proliferation of B-CLL cells (CD5+CD19+), when co-cultured (B-CLL: Teff, 1:20 ratio) (p=0.05). Conclusions In B-cell CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors’ samples. The functional analysis of Treg cells indicates that they can effectively suppress the proliferation of T effector cells. Moreover, T effector cells seem to suppress the proliferation of B-CLL cells, while NB cells increase the proliferation of Treg cells. These observations could probably indicate that at the early stages of the disease, where NB cells are more aberrant, Treg cells’ activity is induced, leading to Teff cells’ suppression and therefore, to an indirect induction of B-CLL cells’ proliferation. Disclosures: No relevant conflicts of interest to declare.

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