scholarly journals Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

2000 ◽  
Vol 350 (3) ◽  
pp. 723-734 ◽  
Author(s):  
Christine LACABARATZ-PORRET ◽  
Sophie LAUNAY ◽  
Elisabeth CORVAZIER ◽  
Raymonde BREDOUX ◽  
Béla PAPP ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Western Blotting ◽  
Time Course ◽  
In Vitro Study ◽  
Fold Increase ◽  
Distinct Pattern ◽  
Platelet Glycoprotein ◽  
Protein Patterns ◽  
Fold Induction

The endoplasmic reticulum (ER) plays a key role in Ca2+ signalling through Ca2+ release via inositol 1,4,5-trisphosphate receptors (InsP3-Rs) and Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a–c RNAs and InsP3-Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose–response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10-8 M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that InsP3-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP3-R types in PMA-induced MEG 01 cells revealed that: (i) InsP3-RI protein and mRNA showed no changes; (ii) InsP3-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP3-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP3-R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca2+-dependent platelet functions.

scholarly journals Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Noelle Zurmühl ◽  
Anna Schmitt ◽  
Ulrike Formentini ◽  
Johannes Weiss ◽  
Heike Appel ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Healthy Subjects ◽  
Time Course ◽  
In Vitro Study ◽  
Allergic Diseases ◽  
Plasmacytoid Dendritic Cells ◽  
Antigen Uptake ◽  
Der P 1 ◽  
The Impact

Abstract Background Human plasmacytoid dendritic cells (pDC) have a dual role as interferon-producing and antigen-presenting cells. Their relevance for allergic diseases is controversial. and the impact of pDC on allergic immune responses is poorly understood. Methods This in vitro study on human pDC isolated from peripheral blood was designed to compare side by side the uptake of three clinically relevant representative allergens: fluorochrome-labeled house dust mite Der p 1, Bee venom extract from Apis mellifera (Api) and the food allergen OVA analyzed flow cytometry and confocal microscopy. Results We found that the internalization and its regulation by TLR9 ligation was significantly different between allergens in terms of time course and strength of uptake. Api and OVA uptake in pDC of healthy subjects was faster and reached higher levels than Der p 1 uptake. CpG ODN 2006 suppressed OVA uptake and to a lesser extent Der p 1, while Api internalization was not affected. All allergens colocalized with LAMP1 and EEA1, with Api being internalized particularly fast and reaching highest intracellular levels in pDC. Of note, we could not determine any specific differences in antigen uptake in allergic compared with healthy subjects. Conclusions To our knowledge this is the first study that directly compares uptake regulation of clinically relevant inhalative, injective and food allergens in pDC. Our findings may help to explain differences in the onset and severity of allergic reactions as well as in the efficiency of AIT.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

scholarly journals Photoacoustic Spectral Sensing Technique for Diagnosis of Biological Tissue Coagulation: In-Vitro Study

Diagnostics ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 133
Author(s):  
Deblina Biswas ◽  
George C. K. Chen ◽  
Hyoung Won Baac ◽  
Srivathsan Vasudevan
Keyword(s):  
Biological Tissue ◽  
In Vitro Study ◽  
Fold Increase ◽  
Dominant Frequency ◽  
Cancer Tissue ◽  
Thermal Coagulation ◽  
Tissue Heating ◽  
Coagulation Therapy ◽  
Spectral Sensing

Thermal coagulation of abnormal tissues has evolved as a therapeutic technique for different diseases including cancer. Tissue heating beyond 55 °C causes coagulation that leads to cell death. Noninvasive diagnosis of thermally coagulated tissues is pragmatic for performing efficient therapy as well as reducing damage of surrounding healthy tissues. We propose a noninvasive, elasticity-based photoacoustic spectral sensing technique for differentiating normal and coagulated tissues. Photoacoustic diagnosis is performed for quantitative differentiation of normal and coagulated excised chicken liver and muscle tissues in vitro by characterizing a dominant frequency of photoacoustic frequency spectrum. Pronounced distinction in the spectral parameter (i.e., dominant frequency) was observed due to change in tissue elastic property. We confirmed nearly two-fold increase in dominant frequencies for the coagulated muscle and liver tissues as compared to the normal ones. A density increase caused by tissue coagulation is clearly reflected in the dominant frequency composition. Experimental results were consistent over five different sample sets, delineating the potential of proposed technique to diagnose biological tissue coagulation and thus monitor thermal coagulation therapy in clinical applications.

Impaired Haemostasis by Intravenous Administration of a Gelatin-based Plasma Expander in Human Subjects

1998 ◽  
Vol 79 (02) ◽  
pp. 286-290 ◽  
Author(s):  
M. Levi ◽  
F. Berends ◽  
A. E. van der Ende ◽  
J. W. ten Cate ◽  
C. P. Stoutenbeek ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Human Subjects ◽  
Fold Increase ◽  
Coagulation Factors ◽  
Platelet Glycoprotein ◽  
Plasma Expander ◽  
Plasma Substitute ◽  
Significant Impairment ◽  
Randomised Controlled

SummaryThe aim of this study was to investigate the effects of a gelatin-based plasma expander on blood coagulation and haemostasis in human subjects.Six healthy men were studied in a randomised, controlled cross-over study to investigate the effects of a 60 min intravenous infusion of either 1 l gelatin-based plasma substitute (Gelofusine) or 0.9% NaCl (control). The infusion of gelatin resulted in a 1.7 fold increase in bleeding time at 60 min and a 1.4 fold increase at 120 min, while saline had no effect (p <0.05). Aggregation studies revealed a significant impairment of ristocetin-induced platelet aggregation (p <0.05), associated with a substantial decrease of vWF:ag (–32% vs. –5%, p <0.05) and ristocetin co-factor (–29% vs. +1%, p <0.05) and without in vitro impairment of the platelet glycoprotein 1b receptor. Gelatin caused a decrease in thrombin-antithrombin complexes (–45% vs. –4%, p <0.05) and F1+2 (–40% vs. +1%, p <0.05). The decrease in circulating levels of vWF:ag, vWF R:Co, thrombin-antithrombin complexes and F1+ 2 was more than could be expected by the calculated plasma-dilution generated by Gelofusine.Our results demonstrated that the administration of a gelatin-based plasma substitute results in a significant impairment of primary haemostasis and thrombin generation. The defect in primary haemostasis appears to be related to a gelatin-induced reduction in von Willebrand factor, whereas the decreased thrombin generation may be due to the dilution of coagulation factors induced by Gelofusine.

In Vitro Analyses of Umbilical Cord Blood (UCB) Derived Monocytes Support Adjunct Topical Application to Augment Platelet Rich Plasma (PRP) in Wound Healing.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1263-1263 ◽  
Author(s):  
Graham C. Chapman ◽  
Nicholas Greco ◽  
Richard Patrick Weitzel ◽  
Phil Paul ◽  
Peter Haviernik ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Topical Application ◽  
Time Course ◽  
Basal Medium ◽  
Fold Increase ◽  
Conditioned Media ◽  
Tubule Formation ◽  
Cell Networks

Abstract Currently, PRP is used clinically as a topical application to augment healing of both surgical and chronic, non-healing wounds. PRP exerts its efficacy by releasing growth factors that enhance clot formation and vasculogenesis. We conducted in vitro functional analyses comparing PRP and/or UCB-derived monocytes including cytokine production, cell migration, and HUVEC tubule formation in standard matrigel assays to test the hypothesis whether topical concurrent application of PRP and UCB-derived monocytes may serve to augment wound healing beyond the ability of topical PRP alone. UCB was obtained according to institutional guidelines and collected into bags with citrate dextrose (Allegiance). MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD14+ monocytes were isolated using AutoMACS magnetic cell sorter (Miltenyi), and cultured in RPMI with 1% HSA. PRP was isolated from adult peripheral blood by centrifugation. To determine if the addition of UCB monocytes may improve the wound healing effects of PRP alone, VEGF, bFGF, and PDGF secreted by monocytes alone, PRP alone, and monocytes supplemented with 3% PRP, were measured by ELISA (RayBiotech) daily over 4 days. PRP alone elicited no measurable secretion of VEGF. UCB-derived monocytes alone showed a low, constant production of VEGF over the four days of 0.868ng/ml. PRP supplemented with UCB-derived monocytes secreted VEGF at a 7.6-fold increase over either PRP or UCB monocytes alone, with a peak production at day three of 6.638ng/ml. PRP alone produced no measurable secretion of bFGF over the four day time course. UCB monocytes alone secreted bFGF in an increasing manner during the same time course. During days one to four, bFGF secreted by UCB monocytes was 33.8, 27.9, 115.4, and 452.1pg/ml, respectively. The presence of PRP suppressed this secretion, as PRP combined with UCB monocytes constantly secreted bFGF at an average of 39.9pg/ml throughout days one to four. Finally, secretion of PDGF was highest in conditions including PRP combined with UCB monocytes. PRP alone constantly produced PDGF at an average of 3,144pg/ml over a 4 day time course. Monocytes alone secreted PDGF constantly at a lower average of 597pg/ml. PRP supplemented with UCB monocytes secreted PDGF at a concentration 5.9-fold higher than PRP alone, producing an average of 18,534pg/ml over four days. To determine whether UCB-derived monocytes respond to cytokines elicited by injured vascular endothelial cells, we measured UCB-derived monocyte chemotaxis to HUVEC conditioned media in hypoxic conditions (5% O2). Migration experiments were conducted using Transwell plates with 8.0 μm pores. Monocytes were cultured in RPMI with 5% FBS at a concentration of 5×106/ml and were allowed to migrate for four hours to either: media alone, PRP, HUVEC-conditioned media, or HUVEC-conditioned media supplemented with PRP. We observed a 3.3 fold increase in the migration of the monocytes to HUVECconditioned media over that of basal media. Experiments with PRP alone showed no significant difference in monocyte migration compared to basal medium. To determine whether UCB-derived monocytes may serve to augment endothelial cell function beyond that elicited by PRP alone, matrigel experiments were conducted by adding HUVEC in endothelial cell basal medium. HUVEC tubule formation in matrigel in basal media was compared in three conditions including media conditioned with: 1) PRP alone, 2) UCB monocytes alone, or 3) a combination of PRP + UCB monocytes. We compared the kinetics and stability of enclosed endothelial cell networks formed by HUVEC. No significant benefit was seen with addition of PRP conditioned media. The number of enclosed endothelial cell networks reached a higher maximum with the addition of monocyte conditioned media (137 networks) as well as PRP + monocyte conditioned media (142 networks), compared to non-conditioned media (80 networks). UCB monocyte and PRP + UCB monocyte conditioned media also improved the stability of the enclosed cell networks in culture as structures persisted beyond 24h, while none were present in the PRP-conditioned or non-conditioned media matrigel cultures. Figure Figure In summary, these in vitro analyses support the hypothesis that UCB-derived monocytes significantly improve efficacy of PRP alone in augmentation of vasculogenesis and cell migration to vascular endothelial injury, thereby supporting potential concurrent topical application of UCB-derived monocytes to PRP in wound healing.

An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

2005 ◽  
Vol 24 (11) ◽  
pp. 573-580 ◽  
Author(s):  
N Mohorko ◽  
N Kregar-Velikonja ◽  
G Repovs ◽  
M Gorensek ◽  
M Bresjanac
Keyword(s):  
Cell Death ◽  
Cell Transplantation ◽  
Fluorescence Intensity ◽  
Time Course ◽  
In Vitro Study ◽  
Host Cells ◽  
Hoechst 33342 ◽  
Overnight Incubation ◽  
Donor Cells

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 mg/mL of H342. H342 labeled / 50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week.Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 mg/mL of H342. Although a 12-hour incubation of rBMSC in 1 mg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.

scholarly journals Zinc and the Zinc Transporter SLC39A10/ZIP10 Are Required for Heme Synthesis in Developing Erythroid Progenitors

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 1320-1320
Author(s):  
Juyoung Kim ◽  
Jaekwon Lee ◽  
Moon-Suhn Ryu
Keyword(s):  
In Vitro Study ◽  
Fold Increase ◽  
Zinc Transporter ◽  
The Body ◽  
Zinc Homeostasis ◽  
Mean Corpuscular Hemoglobin ◽  
Erythroid Progenitors ◽  
Heme Synthesis ◽  
Cellular Zinc

Abstract Objectives Zinc is an essential nutrient for diverse biological processes in the body. Cellular zinc homeostasis is established through differential expressions of the transmembrane zinc transporter proteins, ZnTs and ZIPs. The aims of the current studies were to elucidate the roles of cellular zinc in erythrocyte maturation, and to determine the zinc transporters essential to erythroid zinc homeostasis. Methods G1E-ER4 mouse cells were employed as an in vitro study model of terminal erythroid differentiation. A cell-impermeable zinc chelator, diethylenetriamine pentaacetate (DTPA), was used to limit extracellular zinc availability. For gene silencing, gene-specific siRNAs were introduced to cells via Nucleofection. Functional impacts of zinc and gene deficiency were assessed via ICP-MS-based metal quantitation, heme assays, and gene expression assays using RNA-seq, qPCR, and western analyses. Results G1E-ER4 cells featured a 1.7-fold increase in total cellular zinc contents after 48 h of differentiation. Restriction of zinc import by 50 µM of DTPA led to less red coloration and lower increases in mean corpuscular hemoglobin contents by development. The heme deficiency by DTPA was fully restored by the addition of equimolar zinc, and was not due to changes in cellular iron contents. Zinc-deficient G1E-ER4 cells differentiated with normal Alas2 and Hbb-b1 transcript responses, but less Alad and alpha-globin expressions. Among the 24 zinc transporter genes, Zip10 produced the most prominent response to zinc restriction in differentiating erythroid cells. ZIP10-deficient G1E-ER4 cells were less efficient than control cells in hemoglobin production under zinc restriction. ZIP10 deficiency alone had no effects on the molecular indices of red cell hemoglobinization. Conclusions Our studies characterize zinc as a nutrient essential to normal erythroid maturation and heme synthesis. Moreover, we have identified a compensatory role of ZIP10 for erythroid zinc homeostasis during zinc restriction. Thus, poor zinc status and ZIP10 mutations might serve as potential risk factors and thus new therapeutic targets for anemia and other erythrocyte-related disorders. Funding Sources Supported by CFANS Graduate Fellowship to JK, and the Allen Foundation, Inc. and USDA NIFA Hatch Funds to M-SR.

scholarly journals Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential

2021 ◽  
Vol 22 (16) ◽  
pp. 8764
Author(s):  
Stefano Pulcini ◽  
Lucia Merolle ◽  
Chiara Marraccini ◽  
Eleonora Quartieri ◽  
Daniele Mori ◽  
...  
Keyword(s):  
Growth Factors ◽  
Platelet Rich Plasma ◽  
In Vitro Study ◽  
Fold Increase ◽  
Positive Control ◽  
Osteogenic Potential ◽  
Standardized Protocol ◽  
Proliferation And Differentiation ◽  
Potential Methods

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

scholarly journals Cell-penetrating doxorubicin released from Elastin-like polypeptide kills doxorubicin-resistant cancer cells in vitro study

2020 ◽  
Author(s):  
Jung Su Ryu ◽  
Felix Kratz ◽  
Drazen Raucher
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
In Vitro Study ◽  
Fold Increase ◽  
Membrane Penetration ◽  
Cancer Cell Death ◽  
Cell Penetrating ◽  
Resistant Cells

Abstract Background: Elastin-like polypeptide (ELP) undergoes its characteristic of phase transitioning in response to ambient temperature. ELP therefore has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be targeted to hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer, with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of ELP, a matrix metalloproteinase (MMP) substrate, a cell penetrating peptide (CPP), and 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This construct may be initially targeted to the tumor by application of mild heat after administration. Within the hyperthermic tumor, then this construct is cleaved by MMP, releasing CPP-Dox, which can infiltrate tumor tissues and penetrate cell membranes.Methods: We produced the construct in E.coli and examined its cleavage by MMP enzymes in vitro. Flow cytometry and confocal analysis were used to verify the facilitated uptake of the digested cell-penetrating Dox by breast cancer cells and Dox-resistant cells. Cytotoxicity tests further demonstrated improvements in bioavailability of cell-penetrating Dox following the enhanced cellular uptake of the cancer cells. Comparisons with the non-cleavable ELP counterpart were paralleled.Results: This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells than the ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES/ADR), ELP-released, cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells than ELP-Dox and free Dox. Conclusion: MMP-digested CPP-Dox shows better membrane penetration and induces more cancer cell death in vitro. This CPP-complexed Dox released from ELP kills even dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

scholarly journals Bovine gamma/delta T cells are stimulated by bovine coronavirus antigens identified by a soluble T cells receptor

2017 ◽  
Vol 73 (7) ◽  
pp. 412-417
Author(s):  
Felix N. Toka
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Γδ T Cells ◽  
In Vitro Study ◽  
Fold Increase ◽  
In Vitro Assays ◽  
Gamma Delta ◽  
Bovine Coronavirus ◽  
Gm Csf

Gamma/delta (γδ) T cells in cattle account for an abundant T cell population. However, little is known regarding the function of γδ T cells as immune cells compared to αβ T cells. Not many pathogen-related antigens have been defined and known to stimulate γδ T cells. To address this information gap, we constructed a soluble receptor for bovine γδ T cells (sγδTCR) that was later used to identify two proteins (156 kDa and 102 kDa) or protein fragments expressed by bovine coronavirus (BCov). The molecular weight of the larger protein suggests it could be the spike glycoprotein of BCov. Subsequently, we used the identified viral proteins to study the reactivity of bovine γδ T cells. In vitro assays showed that purified preparations of the two proteins stimulated WC1+ γδ T cells isolated from cattle. A 4-fold increase in IFN production and a significantly higher expression of MHC class II was observed. However, these viral ligands could not stimulate γδ T cells to synthesize IL-8 or GM-CSF, known to be produced by γδ T cells when stimulated with bacterial antigens. Although the γδ T cells assessed here appeared activated by way of IFN and MHC II expression, surface markers such as CD2, CD25, CD44, CD62L and CD335 were not expressed at significant levels. Further, the activation elicited by viral ligands was not sufficient to induce cytotoxic capability in γδ T cells in vitro as measured by a flow cytometry-based cytotoxicity assay. This in vitro study shows that WC1+ γδ T cells can directly recognize viral antigen

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