scholarly journals Analysis of lymphocyte subsets of bone marrow in patients with rheumatoid arthritis by two colour immunofluorescence and flow cytometry.

1990 ◽  
Vol 49 (3) ◽  
pp. 168-171 ◽  
Author(s):  
M Doita ◽  
S Maeda ◽  
K Kawai ◽  
K Hirohata ◽  
T Sugiyama
Keyword(s):  
Rheumatoid Arthritis ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymphocyte Subsets

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5195-5195
Author(s):  
Lulu Lu ◽  
Yongping Song ◽  
Baogen Ma ◽  
Xiongpeng Zhu ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Lymphocyte Subsets ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

scholarly journals Lymphocyte subsets in normal bone marrow

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1600-1606 ◽  
Author(s):  
P Clark ◽  
DE Normansell ◽  
DJ Innes ◽  
CE Hess
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Lymphocyte Subsets ◽  
Cell Loss ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Myeloid Antigens

Abstract Bone marrow aspirates and biopsies from ten normal donors were stained directly with monoclonal antibodies specific for lymphocyte, monocyte, and myeloid antigens, and were analyzed by flow cytometry. To avoid cell loss, lymphocytes were not specifically isolated prior to staining. T cells comprised 46% of aspirate lymphocytes and 22% of biopsy lymphocytes. Further, the Leu-3:Leu-2 ratio of bone marrow T cells was below 1.0. B cells comprised 8% to 11% of bone marrow lymphocytes in both aspirates and biopsies, and there was a substantial percentage of cells in the lymphocyte window that was negative for all B and T cell markers. The lymphocyte window had very little myeloid contamination; however, when the myeloid window was examined, staining was greater than 90%.

FRI0372 INCREASED EXPRESSION OF NOTCH RECEPTORS ON OSTEOCLAST PROGENITORS INDUCED BY RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 783.1-783
Author(s):  
M. Filipović ◽  
A. Šućur ◽  
D. Flegar ◽  
Z. Jajić ◽  
M. Ikić Matijašević ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Notch Signaling ◽  
Synovial Tissue ◽  
Peripheral Blood ◽  
Notch 1 ◽  
Notch 3 ◽  
Notch Receptors

Background:Systemic and periarticular bone loss in rheumatoid arthritis (RA) is mediated by osteoclasts, multinucleated cells originating from the myeloid lineage. Recently, Notch signaling pathway has emerged as a potential regulator of osteoclast progenitor (OCP) differentiation and activation.Objectives:The exact role of Notch signaling in the context of arthritis is still unknown; however, its inhibition has beneficial effects in animal arthritis models. We aimed to determine the expression of Notch receptors and ligands on specific OCP subpopulations and define changes that occur in murine collagen-induced arthritis (CIA) and RA patients.Methods:Peripheral blood, synovial tissue and subchondral bone marrow were collected from RA patients, and periarticular bone marrow (PBM) and spleen (SPL) were harvested from male C57/Bl6 mice immunized with chicken type II collagen. Notch 1 to 4 receptor expression on OCPs was analyzed by flow cytometry. Gene expression of Notch receptors/ligands was determined by qPCR from tissues and sorted OCPs. Sorted OCPs were cultured, with addition of MCSF and RANKL, in control, IgG, Jagged (Jag) 1 or Delta (DLL) 1 coated wells. Immunohistochemistry (IHC) for Notch 1 and 2 was performed on sections of murine hind paws. Research was approved by Ethics committee.Results:We previously identified peripheral and periarticular subpopulations of murine and human OCPs, as CD45+CD3-B220-NK1.1-CD11blo/+CD115+CCR2+and CD45+CD3-CD19-CD56-CD11b+CD14+CCR2+respectively, specifically associated with arthritis. Flow cytometry revealed that majority of murine splenic and periarticular OCPs express Notch 2, whereas Notch 1 and 4 were expressed on approximately 10% of cells. In CIA, this highly osteoclastogenic population is expanded as is the expression of Notch 4 in PBM and Notch 3 in SPL. Majority of human peripheral-blood OCPs express Notch 2 and 4, with a specific increase in the expression of Notch 1 and 3 in RA. In contrast, RA synovial-derived OCPs mostly express Notch 1 to 3, whereas subchondral OCPs mostly express Notch 1 and 4. Notch ligands were analyzed at mRNA level and revealed expression of Jag1, Jag2 and DLL4 in murine sorted OCPs and Jag1 and DLL1 in human sorted OCPs. Expression of Notch 1 and 2 was confirmed by IHC on arthritic murine hind paws, with Notch 2 expressed by bone marrow, synovial tissue and chondrocytes and Notch 1 expressed by chondrocytes and synovial tissue. Increased expression of Notch 1, Notch 2 and Jag1 was also confirmed in murine arthritic periarticular tissue by qPCR. During osteoclastogenic culture, murine and human OCPs exhibit a similar gene expression pattern with higher initial expression of Notch 1 and 2, and increase in the expression of Notch 3 and 4 with differentiation. Osteoclasts were also differentiated under Notch-ligand stimulation. Coating with DLL1 results in a greater number of cells expressing osteoclast-specific TRAP, whereas Jag1 seemed to inhibit osteoclastogenesis.Conclusion:Our results indicate that murine and human OCPs express a distinct tissue-specific pattern of Notch receptors. Notch signaling in OCPs is increased in arthritis and may contribute to the osteoclastogenic potential and increased bone resorption. Our next aim would be to determine the effect of Notch inhibition on OCP activity and arthritis severity.Acknowledgments:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, IP-2014-09-7406 and DOK-2018-09-4276.Disclosure of Interests:None declared

scholarly journals Lymphocyte subsets in normal bone marrow

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1600-1606
Author(s):  
P Clark ◽  
DE Normansell ◽  
DJ Innes ◽  
CE Hess
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Lymphocyte Subsets ◽  
Cell Loss ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Myeloid Antigens

Bone marrow aspirates and biopsies from ten normal donors were stained directly with monoclonal antibodies specific for lymphocyte, monocyte, and myeloid antigens, and were analyzed by flow cytometry. To avoid cell loss, lymphocytes were not specifically isolated prior to staining. T cells comprised 46% of aspirate lymphocytes and 22% of biopsy lymphocytes. Further, the Leu-3:Leu-2 ratio of bone marrow T cells was below 1.0. B cells comprised 8% to 11% of bone marrow lymphocytes in both aspirates and biopsies, and there was a substantial percentage of cells in the lymphocyte window that was negative for all B and T cell markers. The lymphocyte window had very little myeloid contamination; however, when the myeloid window was examined, staining was greater than 90%.

scholarly journals Diastolic dysfunction in a pre-clinical model of diabetes is associated with changes in the cardiac non-myocyte cellular composition

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Charles D. Cohen ◽  
Miles J. De Blasio ◽  
Man K. S. Lee ◽  
Gabriella E. Farrugia ◽  
Darnel Prakoso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Diastolic Dysfunction ◽  
Systolic Function ◽  
Cardiac Fibroblasts ◽  
Detailed Examination ◽  
Diabetic Mice ◽  
Cellular Mechanisms ◽  
Clinical Model ◽  
Cardiac Ventricles

Abstract Background Diabetes is associated with a significantly elevated risk of cardiovascular disease and its specific pathophysiology remains unclear. Recent studies have changed our understanding of cardiac cellularity, with cellular changes accompanying diabetes yet to be examined in detail. This study aims to characterise the changes in the cardiac cellular landscape in murine diabetes to identify potential cellular protagonists in the diabetic heart. Methods Diabetes was induced in male FVB/N mice by low-dose streptozotocin and a high-fat diet for 26-weeks. Cardiac function was measured by echocardiography at endpoint. Flow cytometry was performed on cardiac ventricles as well as blood, spleen, and bone-marrow at endpoint from non-diabetic and diabetic mice. To validate flow cytometry results, immunofluorescence staining was conducted on left-ventricles of age-matched mice. Results Mice with diabetes exhibited hyperglycaemia and impaired glucose tolerance at endpoint. Echocardiography revealed reduced E:A and e’:a’ ratios in diabetic mice indicating diastolic dysfunction. Systolic function was not different between the experimental groups. Detailed examination of cardiac cellularity found resident mesenchymal cells (RMCs) were elevated as a result of diabetes, due to a marked increase in cardiac fibroblasts, while smooth muscle cells were reduced in proportion. Moreover, we found increased levels of Ly6Chi monocytes in both the heart and in the blood. Consistent with this, the proportion of bone-marrow haematopoietic stem cells were increased in diabetic mice. Conclusions Murine diabetes results in distinct changes in cardiac cellularity. These changes—in particular increased levels of fibroblasts—offer a framework for understanding how cardiac cellularity changes in diabetes. The results also point to new cellular mechanisms in this context, which may further aid in development of pharmacotherapies to allay the progression of cardiomyopathy associated with diabetes.

Immature platelet values indicate impaired megakaryopoietic activity in neonatal early-onset thrombocytopenia

2010 ◽  
Vol 103 (05) ◽  
pp. 1016-1021 ◽  
Author(s):  
Hannes Hammer ◽  
Christoph Bührer ◽  
Christof Dame ◽  
Malte Cremer ◽  
Andreas Weimann
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Intensive Care ◽  
Early Onset ◽  
Absolute Number ◽  
Severe Thrombocytopenia ◽  
Platelet Counts ◽  
Immature Platelet Fraction ◽  
Neonatal Thrombocytopenia ◽  
Immature Platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

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