Selective Lympholysis: A Unique Method Using the Double-Dye Technique.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3884-3884
Author(s):  
William J. Owens ◽  
Thomas C. Cesario ◽  
Edward Shanbrom
Keyword(s):  
Methylene Blue ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Cell Types ◽  
The Novel ◽  
Treated Sample ◽  
Unique Method ◽  
Series Of Experiments ◽  
Lymphocytic Cell Line

Abstract Many methods are utilized to destroy mononuclear cells (primarily lymphocytes) either for neoplasm or immunosuppression. Neither radiation nor chemotherapy are truly selective or completely successful. The concept of “Double-Dye” treatment of blood for transfusion has been developed in order to inactivate parasites, bacteria and viruses (J Thromb Haemost2003; 1 Supplement 1 July: P1114). In recent studies, it has been observed that this same “Double-Dye” concept presents the possibility of very selectively eliminating lymphocytes (mononuclear cells) without affecting neutrophils in whole blood. To demonstrate the selectivity of dyes for lymphocytic/mononuclear cell types, two sets of experiments were performed. In the first, 0.3% (w/v) of the “Double Dye’ solution was added to several normal citrated whole blood samples to assess the effect on normal cells, compared to an untreated control. At 24hours post treatment, the lymphocyte count in the treated sample had dropped more than 80%, while little effect on neutrophils was noted. The control counts showed little change for either lymphocytes or neutrophils. Table 1. Lymphocyte reduction by 0.3%(w/v) Double-dye solution (units: cells/mm3). 0hr Control 0.3% Dye WBC 6850 ± 560 6920 ± 630 Neut 5030 ± 375 5100 ± 420 Lymph 1485 ± 220 1490 ± 265 24hr Control 0.3% Dye WBC 6700 ± 480 5300 ± 505 Neut 5025 ± 360 5025 ± 420 Lymph 1474 ± 240 275 ± 75 In the second series of experiments, 0.3%(w/v) “Double-Dye” solution or 0.15%(w/v) Crystal Violet or 0.15%(w/v) Methylene Blue were added to two T-cell leukemia lines (Jurkat, L1210), with a non-malignant, non-lymphocytic cell line (WISH) for the control. The combination of dyes showed the most potent activity against the lymphocytic lines, while the control was virtually unaffected. Table 2: Viability of cell lines after 24 hour exposure to dye solutions. Jurkat L1210 WISH Control 100% ± 2% 100% ± 4% 99% ± 2% 0.3% Double Dye 37% ± 5% 12% ± 4% 88% ± 9% 0.15% Meth. Blue 58% ± 12% 52% ± 9% 100% ± 3% 0.15% Cr. Violet 90% ± 12% 92% ± 10% 99% ± 4% The novel use of these dyes reported here coincided with the recent interest in utilizing methylene blue to increase transfusion safety, but recognizing that the concurrent need to photoactivate was too toxic to certain proteins and didn’t inactivate all pathogens (Transfusion2003; 43(9): 1238–47). Studies investigating the in-vivo efficacy of these novel immunosuppressive and chemotherapeutic methods are currently underway.

scholarly journals Peptide Aggregation Induced Immunogenic Rupture (PAIIR)

2021 ◽  
Author(s):  
Gokhan Gunay ◽  
Seren Hamsici ◽  
Handan Acar ◽  
Mark L. Lang ◽  
Gillian A. Lang ◽  
...  
Keyword(s):  
Immune Response ◽  
Membrane Damage ◽  
Cell Types ◽  
Peptide Aggregation ◽  
Vaccine Adjuvants ◽  
Humoral And Cellular Immunity ◽  
Influenza Hemagglutinin ◽  
Series Of Experiments ◽  
Molecular Patterns

Under the influence of stress and membrane damage, cells undergo immunogenic cell death (ICD), which involves the release of damage associated molecular patterns (DAMPs), natural adjuvants for enhancing an immune response. In the presence of an antigen, released DAMPs can determine the type and magnitude of the immune response, and therefore the longevity and efficacy of an antigen-specific immunity. In the last decade, the immune response effect of ICD has been shown, yet there is no tool that can induce controlled ICD with predictable results, regardless of the cell type. We designed a peptide-based tool, called [II], for controlled damage to cell membrane to induce ICD and DAMPs release. Herein we describe a series of experiments that determine that the mechanism of action of [II] includes a caspase-dependent ICD and subsequent release of immune stimulating DAMPs, on various cell types. Moreover, we tested the hypothesis that controlled DAMP release via [II] in vivo was associated with enhancement of antigen-specific adaptive immunity with influenza hemagglutinin (HA) subunit vaccine. HA and [II] showed significantly higher HA specific IgG1 and IgG2a antibodies, compared to HA-only immunized mice, while the peptide itself did not elicit antibodies. In this paper, we demonstrate the first peptide-aggregation induced immunogenic rupture (PAIIR) approach as vaccine adjuvants for increasing both humoral and cellular immunity. In consideration of its ability to enhance IgG2a responses that are associated with heterosubtypic influenza virus protection, PAIIR is a promising adjuvant to promote universal protection upon influenza HA vaccination.

Differential Leukocyte Recruitment From Whole Blood Via Endothelial Adhesion Molecules Under Shear Conditions

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4691-4699 ◽  
Author(s):  
Paul H. Reinhardt ◽  
Paul Kubes
Keyword(s):  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
The Novel ◽  
Vascular Cell Adhesion ◽  
High Affinity ◽  
Relative Shear ◽  
Endothelial Adhesion Molecules ◽  
Molecule Concentration

Abstract The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti–4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.

scholarly journals CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells

2000 ◽  
Vol 74 (18) ◽  
pp. 8550-8557 ◽  
Author(s):  
Gene G. Olinger ◽  
Mohammed Saifuddin ◽  
Gregory T. Spear
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Virus Binding ◽  
Peripheral Blood Mononuclear ◽  
Human Immunodeficiency Virus Strain ◽  
Immunodeficiency Virus

ABSTRACT The ability of human immunodeficiency virus strain MN (HIVMN), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIVMN bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4+) and CD4-negative (CD4−) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4− cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4− cells was not due to stimulatory signals provided by CD4− cells or infection of CD4− cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4−cells is efficiently passed to CD4+ T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4− cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4− cells is an important route for infection of T cells in vivo.

scholarly journals Study on the Effect of the Three-Dimensional Electrode in Degradation of Methylene Blue by Lithium Modified Rectorite

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Jian Huang ◽  
Yin’an Ming ◽  
Ying Du ◽  
Yingru Wang ◽  
Ci’en Wang
Keyword(s):  
Methylene Blue ◽  
Electrical Property ◽  
Adsorption Property ◽  
Low Cost ◽  
Three Dimensional ◽  
Cell Voltage ◽  
Operating Conditions ◽  
The Novel ◽  
Synthetic Solution ◽  
Series Of Experiments

This study presents the electrochemical degradation of methylene blue (MB) wastewater in a synthetic solution using three-dimensional particle electrodes. The novel particle electrodes were fabricated in this work using the lithium modified rectorite (Li-REC). The adsorption property of the fabricated particle electrodes was studied in a series of experiments. The optimum electrochemical operating conditions of plate distance, cell voltage, and concentration of electrolyte were 2 cm, 9 V, and 0.06 mol L−1, respectively. It was also found that microwave irradiation can effectively improve the adsorption property and electrical property of the fabricated electrodes. In addition, the scanning electron microscope (SEM) of the fabricated electrodes was investigated. The experimental results revealed the order of adsorption property and electrical property of the fabricated electrodes. So, fabricated electrodes are not only of low cost and mass produced, but also efficient to achieve decolorization of MB solution.

Inhibition of Bortezomib-Induced Apoptosis in Chronic Lymphocytic Leukemia by Red Blood Cell Uptake.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5027-5027
Author(s):  
Luise M.C. Wheat ◽  
Susan L. Kohlhaas ◽  
Johan Monbaliu ◽  
Roland De Coster ◽  
Aneela Majid ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Cell Uptake ◽  
Reversible Inhibitor ◽  
Apoptotic Pathway ◽  
Mutational Status ◽  
Induced Apoptosis

Abstract Bortezomib (PS-341/Velcade™) is a reversible inhibitor of the proteasome that has shown promising activity in clinical trials in several malignancies including multiple myeloma, mantle cell lymphoma and follicular lymphoma, including those with refractory disease. However, results have been less encouraging in chronic lymphocytic leukemia (CLL) and we have, therefore, sought to determine the barriers to effective therapy with bortezomib in this disease. Patients with CLL were eligible but were required to have received no therapy in the six months prior to the study. In a panel of 26 patients with CLL, both purified mononuclear cells and whole blood were tested for their apoptotic response to bortezomib (1–100 nM) up to 24 h by flow cytometry and western blotting. In all cases, purified CLL cells were sensitive to bortezomib-induced apoptosis in a concentration and time-dependent fashion, irrespective of stage of disease, resistance to prior therapy, IGHV mutational status or the presence of TP53 mutations. Apoptosis was induced at low (>10 nM) nanomolar concentrations of bortezomib by activation of the intrinsic apoptotic pathway. Bortezomib-induced apoptosis correlated with levels of ubiquitination, Bax activation, and caspase cleavage. Apoptosis of CLL cells was obtained at drug levels readily obtained in vivo using currently-used dosing protocols. However, in vitro, it was necessary to maintain these concentrations for 16–24 hours to obtain maximal apoptosis. Apoptosis measured in a whole blood apoptosis assay was markedly less than in isolated lymphocytes at comparable time points and concentrations. Activity of bortezomib in purified cells was not diminished by addition of exogenous plasma but was abrogated by addition of autologous red blood cells (RBC), suggesting preferential active uptake of the drug by these cells. These data were confirmed in animal models showing preferential distribution of bortezomib to the RBC fraction. RBC uptake may therefore account for the low serum levels of bortezomib attained in vivo during terminal half-life and thus the lack of activity against cells in the peripheral blood. Together with pharmacokinetic and in vivo data, these studies suggest that different dosing schedules of bortezomib other than bolus injections may be more effective in patients with CLL.

scholarly journals Oncogene expression in autoimmune and normal peripheral blood mononuclear cells.

1986 ◽  
Vol 163 (5) ◽  
pp. 1292-1307 ◽  
Author(s):  
D M Klinman ◽  
J F Mushinski ◽  
M Honda ◽  
Y Ishigatsubo ◽  
J D Mountz ◽  
...  
Keyword(s):  
B Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Isolated Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Additional Cell ◽  
Normal Controls

PBMC from patients with autoimmune diseases and from normal controls were studied for the expression of several cellular oncogenes. Gene expression was assessed by Northern blot analysis of poly(A)+ RNA obtained from leukapheresis samples. Patients with SLE expressed significantly more c-myc protooncogene RNA than did normal controls. Increased expression of the N-ras protooncogene was found in that subset of patients whose autoimmune disease was very active. Cells from individuals with SLE, but not from those with other autoimmune illnesses, showed significantly decreased levels of the c-myb and c-fos protooncogenes. To examine the implications of these findings, B and T cells were purified from apheresis samples donated by normal volunteers. When mitogen was used to activate the B cells in vitro, their pattern of protooncogene expression changed to resemble that found in freshly isolated cells from lupus patients. These results suggest that the differences detected in the expression of protooncogenes by patients with SLE may be due to the abnormal activation of their B cells in vivo. The pattern of protooncogene expression found in patients with other autoimmune illnesses is consistent with the activation of additional cell types in those diseases.

scholarly journals The Influence of Methylsulfonylmethane on Inflammation-Associated Cytokine Release before and following Strenuous Exercise

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Mariè van der Merwe ◽  
Richard J. Bloomer
Keyword(s):  
Whole Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Knee Extension ◽  
Strenuous Exercise ◽  
Peripheral Blood Mononuclear ◽  
Physically Active ◽  
Inflammatory Molecules

Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties.Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise.Ex vivoandin vitrotesting consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, whilein vivotesting included the evaluation of cytokines before and through 72 hours after exercise.Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM.Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.

scholarly journals MicroRNA levels quantified in whole blood varies from PBMCs

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 183 ◽  
Author(s):  
Sadaf Atarod ◽  
Hannah Smith ◽  
Anne Dickinson ◽  
Xiao-Nong Wang
Keyword(s):  
Whole Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Cell Types ◽  
Microrna Expression ◽  
Peripheral Blood Mononuclear ◽  
Total Rna ◽  
Disease Stages ◽  
Different Cell Types

MicroRNAs are non-coding RNAs that negatively regulate mRNA expression and play significant roles in both health and disease. Differential microRNA expression has been used to aid diagnosis and discriminate disease stages. The accuracy and reliability of microRNA expression measurement is of utmost importance. Quantification of microRNA expression in human peripheral blood is commonly detected using total RNA extracted via different methods. To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs). This study examined miR-146a-5p and miR-155-5p expression using total RNA extracted in parallel from whole blood and PBMCs of 14 healthy volunteers. The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood. Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

scholarly journals MicroRNA levels quantified in whole blood varies from PBMCs

F1000Research ◽  
2015 ◽  
Vol 3 ◽  
pp. 183 ◽  
Author(s):  
Sadaf Atarod ◽  
Hannah Smith ◽  
Anne Dickinson ◽  
Xiao-Nong Wang
Keyword(s):  
Whole Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Cell Types ◽  
Microrna Expression ◽  
Peripheral Blood Mononuclear ◽  
Total Rna ◽  
Disease Stages ◽  
Different Cell Types

MicroRNAs are non-coding RNAs that negatively regulate mRNA expression and play significant roles in both health and disease. Differential microRNA expression has been used to aid diagnosis and discriminate disease stages. The accuracy and reliability of microRNA expression measurement is of utmost importance. Quantification of microRNA expression in human peripheral blood is commonly detected using total RNA extracted via different methods. To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs). This study examined miR-146a-5p and miR-155-5p expression using total RNA extracted in parallel from whole blood and PBMCs of 14 healthy volunteers. The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood. Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

scholarly journals Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe dengue

2018 ◽  
Vol 115 (52) ◽  
pp. E12363-E12369 ◽  
Author(s):  
Fabio Zanini ◽  
Makeda L. Robinson ◽  
Derek Croote ◽  
Malaya Kumar Sahoo ◽  
Ana Maria Sanz ◽  
...  
Keyword(s):  
B Cells ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Dengue Infection ◽  
Cell Types ◽  
Denv Infection ◽  
Molecular Signature ◽  
Severe Dengue ◽  
Peripheral Blood Mononuclear

Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA–containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.

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