scholarly journals Impact of Human Granulocyte and Monocyte Isolation Procedures on Functional Studies

2012 ◽  
Vol 19 (7) ◽  
pp. 1065-1074 ◽  
Author(s):  
Lu Zhou ◽  
Rajesh Somasundaram ◽  
Rosa F. Nederhof ◽  
Gerard Dijkstra ◽  
Klaas Nico Faber ◽  
...  
Keyword(s):  
Positive Selection ◽  
Gradient Centrifugation ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Magnetic Microbeads ◽  
Content Type ◽  
Functional Studies ◽  
Ficoll Density Gradient Centrifugation ◽  
Surface Receptor ◽  
Monocyte Isolation

ABSTRACTOne of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions to the patient genotype has become an important part of contemporary research. Although it is essential to move this field forward, a systematic study comparing the efficacy and suitability for functional studies of the various available protocols for the isolation of these immune cells has not been performed. Here, we compare human granulocyte functionality under three enrichment protocols: (i) Ficoll density gradient centrifugation, (ii) anti-CD15 antibody-conjugated microbeads (positive selection), and (iii) Polymorphoprep. Primary monocytes were isolated in parallel using (i) anti-CD14 magnetic microbeads, (ii) non-monocyte depletion by antibody-conjugated magnetic microbeads (negative selection), (iii) RosetteSep antibody cocktail, and (iv) the classical adherence protocol. The best results in terms of purity and cell functionality were obtained with positive selection by magnetic microbeads for both human granulocytes and monocytes. Whereas phagocytosis ofEscherichia colibacteria was identical in all isolation procedures tested, the granulocyte respiratory burst was higher in positively selected cells. In addition, different granulocyte enrichment procedures affect cell surface receptor expression to different extents.In toto, we propose that positive selection of granulocytes and monocytes be adopted as the procedure of choice for studies of human granulocyte and monocyte functions but caution investigators to be aware of possible alterations in cell phenotypes with different isolation procedures.

scholarly journals Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage

2016 ◽  
Vol 198 (6) ◽  
pp. 994-1004 ◽  
Author(s):  
Arup Dey ◽  
Christopher N. Vassallo ◽  
Austin C. Conklin ◽  
Darshankumar T. Pathak ◽  
Vera Troselj ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kin Recognition ◽  
Myxococcus Xanthus ◽  
Laboratory Strain ◽  
Cell Surface Receptor ◽  
Form Complex ◽  
Content Type ◽  
Recognition Specificity ◽  
Surface Receptor ◽  
Evolutionary Event

ABSTRACTMyxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-typeMyxococcus xanthus(DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large “polyploid prophage,” Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery ofM. xanthusto transfer a toxin that makes the population “addicted” to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (bytraAallele swap) to recognize a closely related species,Myxococcus fulvus. As a result,M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms intraA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes.IMPORTANCEThe transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups.

scholarly journals Porcine Breast Extracellular Matrix Hydrogel for Spatial Tissue Culture

2018 ◽  
Vol 19 (10) ◽  
pp. 2912 ◽  
Author(s):  
Girdhari Rijal ◽  
Jing Wang ◽  
Ilhan Yu ◽  
David Gang ◽  
Roland Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Mammary Epithelial Cell ◽  
Tumor Formation ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Breast Diseases ◽  
Porous Scaffolds ◽  
Ecm Proteins ◽  
Surface Receptor

Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.

scholarly journals Drebrin 1 in dendritic cells regulates phagocytosis and cell surface receptor expression through recycling for efficient antigen presentation

Immunology ◽  
2018 ◽  
Vol 156 (2) ◽  
pp. 136-146 ◽  
Author(s):  
Diana M. Elizondo ◽  
Temesgen E. Andargie ◽  
Naomi L. Haddock ◽  
Thomas A. Boddie ◽  
Michael W. Lipscomb
Keyword(s):  
Dendritic Cells ◽  
Cell Surface ◽  
Antigen Presentation ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Surface Receptor

scholarly journals Selective inhibition of the growth of human erythroid bursts by monoclonal antibodies against transferrin or the transferrin receptor

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1631-1638 ◽  
Author(s):  
KM Shannon ◽  
JW Larrick ◽  
SA Fulcher ◽  
KB Burck ◽  
J Pacely ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transferrin Receptor ◽  
Thymidine Incorporation ◽  
Selective Inhibition ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Transferrin Receptors ◽  
Human Erythropoietin ◽  
Surface Receptor ◽  
Transferrin Receptor Expression

Abstract The relative requirements of colonies derived from erythroid (BFU-E) and myeloid (CFU-c) progenitors for transferrin were examined using monoclonal antibodies directed against the transferrin molecule (TF-6) or its cell surface receptor (TFR-A12, TFR1–2B). Growth of erythroid bursts was profoundly reduced at concentrations of all three antibodies that had no effect on CFU-c-derived colonies. When TFR1–2B was layered over cultures established one to seven days previously, further burst development was inhibited, and degeneration of early erythroid colonies was observed. Addition of erythropoietin augmented transferrin receptor expression on cells harvested after 1 to 2 weeks in culture and analyzed by flow cytometry. Recombinant human erythropoietin gave results comparable to those obtained in experiments using human urinary erythropoietin. Analysis of erythroblasts plucked directly from culture plates confirmed the presence of transferrin receptors on BFU-E-derived colonies. Thymidine incorporation was maximal early in the second week of culture and coincided with high transferrin receptor expression. These data demonstrate that transferrin must be available into the second week of culture to support the growth and differentiation of BFU- E-derived erythroid bursts, that the generation of erythroid colonies from BFU-E is more dependent on transferrin than myeloid colony formation from CFU-c, and that erythropoietin modulates the expression of transferrin receptors on growing bursts.

scholarly journals Identification of the Cellular Receptor of Clostridium spiroforme Toxin

2012 ◽  
Vol 80 (4) ◽  
pp. 1418-1423 ◽  
Author(s):  
Panagiotis Papatheodorou ◽  
Claudia Wilczek ◽  
Thilo Nölke ◽  
Gregor Guttenberg ◽  
Daniel Hornuss ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Receptor ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Content Type ◽  
Recombinant Production ◽  
Host Cell Receptor ◽  
Surface Receptor ◽  
Microscopic Studies ◽  
Host Cell Surface

ABSTRACTClostridium spiroformeproduces the binary actin-ADP-ribosylating toxin CST (C. spiroformetoxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) ofC. spiroformetoxin inBacillus megaterium. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxinsClostridium difficiletransferase (CDT) andClostridium perfringensiota toxin. Microscopic studies revealed that CST, but not the relatedClostridium botulinumC2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR withC. difficileCDT andC. perfringensiota toxin as a host cell surface receptor.

Increased cell-surface receptor expression on U-937 cells induced by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

2000 ◽  
Vol 48 (10) ◽  
pp. 569-578 ◽  
Author(s):  
Marina Y. Pushkareva ◽  
Sharon L. Wannberg ◽  
Andrew S. Janoff ◽  
Eric Mayhew
Keyword(s):  
Cell Surface ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Surface Receptor

scholarly journals Identification of Molecular Phenotypes and Biased Signaling Induced by Naturally Occurring Mutations of the Human Calcium-Sensing Receptor

Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4304-4316 ◽  
Author(s):  
Katie Leach ◽  
Adriel Wen ◽  
Anna E. Davey ◽  
Patrick M. Sexton ◽  
Arthur D Conigrave ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Receptor Coupling ◽  
Calcium Sensing ◽  
Naturally Occurring ◽  
Molecular Determinant ◽  
Biased Signaling ◽  
Surface Receptor ◽  
Molecular Phenotypes

More than 200 naturally occurring mutations have been identified in the human CaSR, which have been linked to diseases involving dysregulation of extracellular Ca2+ homeostasis. These mutations have classically been termed “loss-” or “gain-of-function” mutations, which is an oversimplification given that amino acid changes can alter numerous molecular properties of a receptor. We thus sought to characterize the effects of 21 clinically relevant mutations, the majority located in the heptahelical domains and extracellular loop regions of the CaSR, using flow cytometry to measure cell surface receptor expression levels, and measurements of intracellular Ca2+ mobilization and ERK1/2 phosphorylation to monitor receptor signaling. We identified distinct molecular phenotypes caused by these naturally occurring amino acid substitutions, which included combinations of loss- and gain-of-expression and changes in intrinsic signaling capacity. Importantly, we also identified biased signaling in the response of the CaSR to different mutations across the two pathways, indicating that some mutations resulted in receptor conformations that differentially altered receptor-coupling preferences. These findings have important implications for understanding the causes of diseases linked to the CaSR. A full appreciation of the molecular effects of these amino acid changes may enable the development of therapeutics that specifically target the molecular determinant of impairment in the receptor.

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