4-Aminopyridine activates calcium influx through modulation of the pore-forming purinergic receptor in human peripheral blood mononuclear cells

2004 ◽  
Vol 82 (1) ◽  
pp. 50-56 ◽  
Author(s):  
Ingrid Lajdova ◽  
Dusan Chorvat, Jr. ◽  
Viera Spustova ◽  
Alzbeta Chorvatova
Keyword(s):  
Purinergic Receptors ◽  
Calcium Release ◽  
Mononuclear Cells ◽  
Calcium Influx ◽  
Purinergic Receptor ◽  
Human Peripheral Blood ◽  
Partial Agonist ◽  
Human Lymphocytes ◽  
Calcium Entry ◽  
Peripheral Blood Mononuclear

We investigated whether 4-aminopyridine (4AP), a drug recently linked to calcium influx and apoptosis, also affected purinergic receptor channels that are known to play an important role in the activation of T lymphocytes. The application of 4AP induced a rise in [Ca2+]i that was sensitive to nickel. This action was also observed in cells in which calcium reserves were emptied using thapsigargin (Tg). However, it was not present in the absence of extracellular Ca2+, despite full internal reserves. Adenosine trisphosphate (ATP), a partial agonist and a physiological activator of purinergic receptors, also stimulated Ca2+ entry independently of the calcium release from internal compartments. The effects of 4AP and ATP were not additive when studied on the same population of cells. KN-62 inhibited an increase in calcium entry induced by 4AP, while brilliant blue G (BBG) prevented it, supporting the hypothesis that purinergic P2X7 receptors are involved in this action. Furthermore, 4AP allowed entry of ethidium bromide (314 Da) but not propidium iodide (415 Da) into the cell, also corroborating the involvement of P2X7 pores. The presented results demonstrate, for the first time in human mononuclear cells isolated from healthy volunteers, that the P2X7 channel pore is involved in the action of 4AP and intervenes in the sustained calcium entry induced in response to 4AP.Key words: calcium, human lymphocytes, 4-aminopyridine, purinergic receptors.

scholarly journals Control of hsp70 RNA levels in human lymphocytes.

1987 ◽  
Vol 104 (2) ◽  
pp. 183-187 ◽  
Author(s):  
L Kaczmarek ◽  
B Calabretta ◽  
H T Kao ◽  
N Heintz ◽  
J Nevins ◽  
...  
Keyword(s):  
Culture Medium ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Peripheral Blood Mononuclear ◽  
Growth State ◽  
Steady State Levels ◽  
Rna Blots

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells

1994 ◽  
Vol 14 (5) ◽  
pp. 231-242 ◽  
Author(s):  
Beatrice M. Anner ◽  
Danielle Lacotte ◽  
Rolf M. Anner ◽  
Marlis Moosmayer
Keyword(s):  
Cell Viability ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Exchange Process ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Isolated Human Lymphocytes

A ligand for the digitalis receptor located on the membrane-embedded Na,K-ATPase (NKA; EC 3.6.1.37) has been isolated from bovine hypothalamus (hypothalamic inhibitory factor; HIF) and identified as isomeric ouabain (Tymiaket al, 1993,Proc. Natl. Acad. Sci.90: 8189–8193). In analogy to cardioactive steroids (CS) derived from plants or from toad, HIF inhibits the Na/K-exchange process and the ATPase activity of isolated Na,K-ATPase although by a different molecular action mechanism. In the present work we show that, as plant-derived ouabain, HIF inhibits86Rb-uptake by isolated human lymphocytes with an IC50 of about 20 nM; above this concentration HIF reduces cell viability in contrast to ouabain. The decrease in cell viability by excess HIF is accompanied by discrete morphological alterations (mitochondrial swelling) visible by transmission electron microscopy of ultra-thin sectioned peripheral blood mononuclear cells. Taken together the results show that the hypothalamic NKA inhibitor blocks NKA of isolated human lymphocytes with high potency at nanomolar concentrations without toxicity; concentrations exceeding the ones required to block86Rb-uptake reduce cell viability, probably due to leak formation across the NKA molecule. Thus, lymphocytes constitute a potential target for HIF action and by their altered NKA status a possible messenger between the nervous and the immune system.

scholarly journals Sildenafil does not affect the proliferation of human lymphocytes in the in vitro transplant model

2019 ◽  
Author(s):  
Beata Kaleta
Keyword(s):  
Bone Marrow ◽  
Immune System ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Venous Blood ◽  
Human Immune System ◽  
Pde5 Inhibitors ◽  
Trypan Blue Exclusion ◽  
Peripheral Blood Mononuclear

Sildenafil is a selective inhibitor of type 5 phosphodiesterase (PDE5) used in the treatment of erectile dysfunction (ED) and pulmonary arterial hypertension (PAH). Numerous studies revealed beneficial effects of sildenafil use in chronic kidney disease, and also in renal, liver, heart and bone marrow transplant recipients. Some reports suggest that sildenafil modulates function of the immune system, and additionally proliferation of endothelial, bone marrow and cancer cells. Despite the fact that PDE5 inhibitors showed efficacy and safety, it is very important to know whether these drugs have immunomodulatory properties, because are used in patients after organ transplantation. The influence of sildenafil on antigen-induced proliferation of lymphocytes remains currently unknown, thus the aim of the study was to investigate the effects of the drug on human peripheral blood mononuclear cells (PBMCs) proliferation in a mixed lymphocyte reaction (MLR).PBMCs were isolated from venous blood from 30 donors. The proliferation was examined on the DNA synthesis level by measurements of 3H-thymidine incorporation. Cell viability was determined using trypan blue exclusion method.The study demonstated that sildenafil at concentrations of 0.06 µM, 0.6 µM and 6µM did not affect auto- and alloantigen-induced  proliferation of PBMCs and showed no cytotoxic effect. However, further analysis is required to fully understand the role of PDE5 inhibitors in the regulation of human immune system.

Heligmosomoides polygyrus superantigen: differential response with mouse and human lymphocytes

Parasitology ◽  
1997 ◽  
Vol 115 (5) ◽  
pp. 531-536 ◽  
Author(s):  
M. ROBINSON ◽  
T. R. GUSTAD
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Secretory Proteins ◽  
Cellular Mechanisms ◽  
Peripheral Blood Mononuclear ◽  
Heligmosomoides Polygyrus ◽  
Human Volunteers ◽  
Antigen Presenting ◽  
Proliferation Assays

The interaction between Heligmosomoides polygyrus superantigen and human peripheral blood mononuclear cells (PBMC) was evaluated. Parasite homogenate and excretory–secretory proteins from both L4 larvae and adult worms were examined for their ability to stimulate, and be presented by, human cells. Proliferation assays using PBMC from 3 human volunteers indicated that naïve cells were stimulated by H. polygyrus superantigen. Antigen presenting cells (APC) from a number of human donors were able to successfully present H. polygyrus superantigen to mouse T cell hybridomas. However, this ability varied according to the source of the superantigen, and human APC, in contrast to APC from mice, could only present the superantigen contained within parasite homogenate. Also, in contrast to the situation with mice, human APC could present H. polygyrus superantigen to stimulate mouse T cells expressing not only TCR Vβ8.1, but also TCR Vβ7. Therefore, H. polygyrus superantigen can successfully stimulate, and be presented by, human PBMC of different MHC haplotypes, although the cellular mechanisms appear to be different from those observed in the mouse.

scholarly journals ZERUMBONE, A NATURAL PLANT DIETARY COMPOUND INDUCES EXPRESSION OF INTERLEUKIN-12P70 CYTOKINE IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

2016 ◽  
Vol 9 (9) ◽  
pp. 312 ◽  
Author(s):  
Swagatika Dash ◽  
Monalisa Ray ◽  
Abtar Mishra ◽  
Sajad Shahbazi ◽  
Gopinath Achary K ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Immunomodulatory Activity ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

ABSTRACTObjective: Despite possessing many biological activities as antiproliferative, antioxidant, anti-inflammatory, and anticancerous, and zerumbone lacksany evidence for its immunomodulatory activity. This naturally occurring dietary compound needs to be developed as drug to support therapeuticclaims in various infections and diseases.Methods: Hence, in this study, the immunomodulatory effects of zerumbone were investigated by evaluating the effect of this compound toward thelymphocytes proliferation in human peripheral blood mononuclear cells.Results: Lymphocyte proliferation assay showed that zerumbone was able to activate human lymphocytes at dosage-dependent manner at the highestconcentration 40 μl/mL. The production of human interleukin-12p70 cytokine in culture supernatant from activated lymphocytes was upregulatedby zerumbone at 24 hrs and gradually decreased at 48 hrs. Hence, the study confirms the immunomodulatory activity of zerumbone which play animportant role in boosting up the immune system through cytokine production in dosage dependent manner.Conclusion: The study concludes that zerumbone could be used as a lead molecule in herbal therapeutic world as an immunomodulatory drug in thetreatment of chronic infections and various autoimmune disorders.Keywords: Zerumbone, Peripheral blood mononuclear cells, Immunomodulation, Cytokine, Lymphocyte proliferation.

scholarly journals A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes

2018 ◽  
Vol 123 ◽  
Author(s):  
Lili Wang ◽  
Adolfas K. Gaigalas ◽  
Paul C. DeRose
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Equilibrium Concentration ◽  
Peripheral Blood Mononuclear ◽  
Binding Condition ◽  
Pbmc Sample ◽  
Quantitative Flow

The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources. Four binding conditions were performed, each with the same PBMC sample and different CD4 mAb conjugate. Each binding condition consisted of the PBMC sample incubated for 30 min in labeling solutions containing progressively larger concentrations of the CD4 mAb-label conjugate. After the incubation period, the cells were re-suspended in PBS-based buffer and analyzed using a flow cytometer to measure the mean fluorescence intensity (MFI) of the labeled cell populations. A model was developed to estimate the equilibrium concentration of bound CD4 mAb-label conjugates to CD4 receptors on PBMC. A set of parameters was obtained from the best fit of the model to the measured MFI data and the known number of CD4 receptors on PBMC surface. Divalent and monovalent binding had to be invoked for the APC and FITC CD4 mAb conjugates, respectively. This suggests that the mAb binding depends on the size of the label, which has significant implications for quantitative flow cytometry. The study supports the National Institute of Standards and Technology program to develop quantitative flow cytometry measurements.

scholarly journals Purification and partial characterization of a high hemagglutinating chitin-binding lectin from Aponogeton natans tubers

2021 ◽  
Author(s):  
Shashikanth Dara ◽  
Harikrishna Naik Lavudi ◽  
Venkateswara Rao ◽  
Nanibabu Badithi ◽  
Seshagirirao Kottapalli
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Single Step ◽  
List Type ◽  
Peripheral Blood Mononuclear ◽  
Binding Lectin

AbstractA novel chitin-binding lectin was isolated from the tubers of a plant Aponogeton natans from the monocot family Aponogetonaceae, designated as ANTL (Aponogeton natans tuber lectin). The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes, as well as human blood cells of groups A, B and O with different specificities. Lectin activity is inhibited by the oligomers of N-acetylglucosamine. ANTL is a dimeric glycoprotein with molecular weight of ∼66 KDa and has two identical sub-units of 33 KDa. The carbohydrate percent is 8.2% of the total lectin. The lectin was thermo stable up to 50°C with broad pH optima (pH 4–10). ANTL is found to be potent mitogen for normal murine and human lymphocytes at the concentration as low as 1 µg/ml. Cytotoxic studies of the lectin on human U 266 cell lines has revealed that there is 50% decrease in the proliferation. The confirmation of both the hemagglutination and mitogenic proliferation activity suggests that ANTL is a Chitin-binding lectin with diverse functions. The pharmacological relevance of ANTL as a potent mitogen with some cytotoxic effect in certain cell lines are reported for the first time.HighlightsA novel chitin-binding lectin was purified from the tuber extracts of Aponogeton natans (ANTL) in a single step on chitin column by affinity chromatography.ANTL is a dimeric glycoprotein with a molecular weight of ∼66 KDa with high hemagglutination activity towards rabbit erythrocytes.The cytotoxic effect of ATNL on human cell lines U266 has shown 50 % inhibition of their proliferation.ANTL displayed potent mitogenic response towards murine and human peripheral blood mononuclear cells.

scholarly journals Infection of human lymphomononuclear cells by SARS-CoV-2

2020 ◽  
Author(s):  
Marjorie C Pontelli ◽  
Italo A Castro ◽  
Ronaldo B Martins ◽  
Flávio P Veras ◽  
Leonardo La Serra ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Severe Infection ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
B And T Lymphocytes

AbstractAlthough SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.

A voltage-operable current is involved in Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role

1996 ◽  
Vol 271 (5) ◽  
pp. C1494-C1503 ◽  
Author(s):  
J. J. Densmore ◽  
D. M. Haverstick ◽  
G. Szabo ◽  
L. S. Gray
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Interleukin 2 ◽  
Nordihydroguaiaretic Acid ◽  
Jurkat Cells ◽  
Jurkat T Cells ◽  
Peripheral Blood Mononuclear ◽  
Voltage Gated

Presently, it is thought that a non-voltage-gated current is responsible for activation-induced Ca2+ entry in nonelectrically excitable cells such as lymphocytes. However, it has also been proposed that the pathway instead involves a second messenger-regulated Ca2+ channel that is voltage operable, where "voltage operable" is defined as an intrinsic property of the channel protein(s) rather than a requirement of normal gating. To evaluate the contribution of these currents to activation-induced Ca2+ influx, each was examined with respect to its ability to account for Ca2+ influx as reported by Ca(2+)-sensitive dyes. We identified a set of reagents, nordihydroguaiaretic acid and various calmodulin inhibitors, that inhibits Ca2+ entry and blocks the voltage-operable current but leaves the non-voltage-gated current unaltered. Further-more, nordihydroguaiaretic acid inhibited Ca(2+)-dependent proliferation of mitogen-activated human peripheral blood mononuclear cells or Jurkat T cells and specifically blocked Ca(2+)-dependent interleukin 2 production by Jurkat T cells to a degree similar to the immunosuppressant drug cyclosporin A. We also identified compounds, amiloride and Mn2+, that block the non-voltage-gated current but have no effect on either the voltage-operable current or Ca2+ entry. Correspondingly, amiloride had no effect on Ca(2+)-dependent proliferation of Jurkat cells. These observations imply that blockade of the non-voltage-gated current does not block either Ca2+ entry or Ca(2+)-dependent lymphocyte proliferation, whereas blockade of the voltage-operable current does. The data suggest that the voltage-operable current may be a mediator of activation-induced Ca2+ entry in lymphocytes.

Differential promoter utilization by the c-myc gene in mitogen- and interleukin-2-stimulated human lymphocytes

1987 ◽  
Vol 7 (8) ◽  
pp. 2988-2993
Author(s):  
H E Broome ◽  
J C Reed ◽  
E P Godillot ◽  
R G Hoover
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Mitogen Stimulation ◽  
Blood Mononuclear Cells ◽  
Myc Gene

Transcription of the c-myc gene is initiated from two principal promoters, P1 and P2. We demonstrate here that a shift in promoter utilization occurred with time in human peripheral blood mononuclear cells (PBMC) that had been stimulated to proliferate. The P1/P2 ratio reached a maximum of approximately 1.3 at 4 h after phytohemagglutinin stimulation and a minimum of 0.31 at 48 h. Actinomycin decay experiments demonstrated that both P1 and P2 transcripts had similar half-lives at early and late times after mitogen stimulation, indicating that the shift in promoter utilization was probably not posttranscriptionally regulated. Addition of interleukin-2 to previously activated PBMC increased c-myc mRNA, but unlike increases after mitogen stimulation, the P1/P2 ratio stayed less than 0.5. Our findings demonstrated that there was a difference between mitogen- and interleukin-2-stimulated increases in c-myc RNA in PBMC.

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