scholarly journals Extracellular Vesicles Tropism: A Comparative Study between Passive Innate Tropism and the Active Engineered Targeting Capability of Lymphocyte-Derived EVs

Membranes ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 886
Author(s):  
Tania Limongi ◽  
Francesca Susa ◽  
Bianca Dumontel ◽  
Luisa Racca ◽  
Michela Perrone Donnorso ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
B Lymphocyte ◽  
Fluorescent Microscopy ◽  
Cellular Membrane ◽  
Extracellular Vesicle ◽  
Parental Cell ◽  
Leukemic Cells ◽  
Appreciable Amount ◽  
Phenotypic Characterization ◽  
Parental Cell Line

Cellular communications take place thanks to a well-connected network of chemical–physical signals, biomolecules, growth factors, and vesicular messengers that travel inside or between cells. A deep knowledge of the extracellular vesicle (EV) system allows for a better understanding of the whole series of phenomena responsible for cell proliferation and death. To this purpose, here, a thorough immuno-phenotypic characterization of B-cell EV membranes is presented. Furthermore, the cellular membrane of B lymphocytes, Burkitt lymphoma, and human myeloid leukemic cells were characterized through cytofluorimetry assays and fluorescent microscopy analysis. Through cytotoxicity and internalization tests, the tropism of B lymphocyte-derived EVs was investigated toward the parental cell line and two different cancer cell lines. In this study, an innate capability of passive targeting of the native EVs was distinguished from the active targeting capability of monoclonal antibody-engineered EVs, able to selectively drive the vesicles, enhancing their internalization into the target cancer cells. In particular, the specific targeting ability of anti-CD20 engineered EVs towards Daudi cells, highly expressing CD20 marker on their cell membrane, was proved, while almost no internalization events were observed in HL60 cells, since they did not express an appreciable amount of the CD20 marker on their plasma membranes.

scholarly journals Changes of Circulating Extracellular Vesicles from the Liver after Roux-en-Y Bariatric Surgery

2019 ◽  
Vol 20 (9) ◽  
pp. 2153 ◽  
Author(s):  
Gersina Rega-Kaun ◽  
Dorothea Ritzel ◽  
Christoph Kaun ◽  
Benjamin Ebenbauer ◽  
Barbara Thaler ◽  
...  
Keyword(s):  
Bariatric Surgery ◽  
Extracellular Vesicles ◽  
Gastric Bypass Surgery ◽  
Cellular Membrane ◽  
Extracellular Vesicle ◽  
Parental Cell ◽  
Phospholipid Bilayer ◽  
Cellular Origin ◽  
Specific Proteins ◽  
Hepatic Stress

Circulating extracellular vesicles are small particles enclosed by a phospholipid bilayer. Vesicles deriving directly from the cellular membrane by an active budding process retain cell origin specific proteins and RNA. These vesicles carry pathophysiological information from their parental cell and hold the potential to allow analysis of organs without the need for a biopsy. We included in our study 27 patients undergoing bariatric surgery. Hepatic extracellular vesicles were determined by flow cytometry. mRNA specific for hepatic cellular origin was determined in the extracellular vesicle fraction using qPCR. Surgery led to a massive reduction of weight and overall hepatic stress as determined by alanine transaminase (ALT), aspartate transaminase (AST) and γ-glutamyltransferase (GGT). Total extracellular vesicle numbers were reduced after bariatric surgery. Liver specific vesicles identified by HepPar1 or asialoglycoprotein receptor (ASGPR) were significantly reduced after bariatric surgery in both AnnexinV+ and AnnexinV− subgroups. When analyzing circulating liver-specific mRNAs, we found reduced levels of these mRNAs after surgery even though total circulating RNA remained unchanged. We conclude that circulating hepatic extracellular vesicles are detectable in samples from patients undergoing gastric bypass surgery. These vesicles are reduced after a reduction of hepatic stress also observed with classic liver enzyme measurements. We conclude that ASGPR or HepPar positive vesicles hold the potential to serve as liver specific vesicle markers.

The Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine Treatment in Resistant 2D and 3D Model Triple Negative Breast Cancer Cell Line: ABCG2 Expression Data

2021 ◽  
Vol 21 ◽  
Author(s):  
Fatma Kubra Ata ◽  
Serap Yalcin
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profile ◽  
Three Dimensional ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Xtt Assay ◽  
Gene And Protein Expression

Background: Chemotherapeutics have been commonly used in cancer treatment. Objective: In this study, the effects of Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine have been evaluated on two-dimensional (2D) (sensitive and resistance) cell lines and three dimensional (3D) spheroid structure of MDA-MB-231. The 2D cell culture lacks a natural tissue-like structural so, using 3D cell culture has an important role in the development of effective drug testing models. Furthermore, we analyzed the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene and protein expression profile in this study. We aimed to establish a 3D breast cancer model that can mimic the in vivo 3D breast cancer microenvironment. Methods: The 3D spheroid structures were multiplied (globally) using the three-dimensional hanging drop method. The cultures of the parental cell line MDA-MB-231 served as the controls. After adding the drugs in different amounts we observed a clear and well-differentiated spheroid formation for 24 h. The viability and proliferation capacity of 2D (sensitive and resistant) cell lines and 3D spheroid cell treatment were assessed by the XTT assay. Results: Cisplatin, Irinotecan, 5-Fu, and Gemcitabine-resistant MDA-MB-231 cells were observed to begin to disintegrate in a three-dimensional clustered structure at 24 hours. Additionally, RT-PCR and protein assay showed overexpression of ABCG2 when compared to the parental cell line. Moreover, MDA-MB-231 cells grown in 3D showed decreased sensitivity to chemotherapeutics treatment. Conclusion: More resistance to chemotherapeutics and altered gene expression profile was shown in 3D cell cultures when compared with the 2D cells. These results might play an important role to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR in the 3D spheroid forms.

Regulation of c-met expression in B16 murine melanoma cells by melanocyte stimulating hormone

1999 ◽  
Vol 112 (5) ◽  
pp. 623-630
Author(s):  
D. Rusciano ◽  
P. Lorenzoni ◽  
M.M. Burger
Keyword(s):  
Melanoma Cells ◽  
Parental Cell ◽  
Melanocortin Receptor ◽  
Parental Cell Line ◽  
Melanocyte Stimulating Hormone ◽  
Murine Melanoma ◽  
The One ◽  
Murine Melanoma Cells ◽  
Stimulating Hormone

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(α) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

Insulin-stimulated trafficking of ENaC in renal cells requires PI 3-kinase activity

2003 ◽  
Vol 284 (6) ◽  
pp. C1645-C1653 ◽  
Author(s):  
Bonnie L. Blazer-Yost ◽  
Michail A. Esterman ◽  
Chris J. Vlahos
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Apical Cell ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Insulin Stimulation ◽  
Α Subunit ◽  
Clonal Lines ◽  
Green Fluorescent ◽  
Apical Membranes

αENaC-EGFP (enhanced green fluorescent protein-tagged α-subunit of the epithelial Na+ channel) stably transfected clonal lines derived from the A6 parental cell line were used to study the physical mechanisms of insulin-stimulated Na+ transport. Within 1 min of insulin stimulation, ENaC migrates from a diffuse cytoplasmic localization to the apical and lateral membranes. Concurrently, after insulin stimulation, phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC on the lateral but not apical membrane. An inhibitor of PI 3-kinase, LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but does alter the intracellular site of the colocalization, preventing the translocation of ENaC to the lateral and apical membranes. These data show that insulin stimulation causes the migration of ENaC to the lateral and apical cell membranes and that this trafficking is dependent on PI 3-kinase activity.

scholarly journals Interaction of drugs with lipid raft membrane domains as a possible target

2020 ◽  
Vol 14 (1) ◽  
pp. 34-47
Author(s):  
Hironori Tsuchiya ◽  
Maki Mizogami
Keyword(s):  
Membrane Fluidity ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Plasma Membranes ◽  
Cellular Membrane ◽  
Membrane Lipid ◽  
Membrane Domains ◽  
Functional Protein ◽  
Lipid Complex ◽  
Physicochemical Changes

Introduction: Plasma membranes are not the homogeneous bilayers of uniformly distributed lipids but the lipid complex with laterally separated lipid raft membrane domains, which provide receptor, ion channel and enzyme proteins with a platform. The aim of this article is to review the mechanistic interaction of drugs with membrane lipid rafts and address the question whether drugs induce physicochemical changes in raft-constituting and raft-surrounding membranes. Methods: Literature searches of PubMed/MEDLINE and Google Scholar databases from 2000 to 2020 were conducted to include articles published in English in internationally recognized journals. Collected articles were independently reviewed by title, abstract and text for relevance. Results: The literature search indicated that pharmacologically diverse drugs interact with raft model membranes and cellular membrane lipid rafts. They could physicochemically modify functional protein-localizing membrane lipid rafts and the membranes surrounding such domains, affecting the raft organizational integrity with the resultant exhibition of pharmacological activity. Raft-acting drugs were characterized as ones to decrease membrane fluidity, induce liquid-ordered phase or order plasma membranes, leading to lipid raft formation; and ones to increase membrane fluidity, induce liquid-disordered phase or reduce phase transition temperature, leading to lipid raft disruption. Conclusion: Targeting lipid raft membrane domains would open a new way for drug design and development. Since angiotensin-converting enzyme 2 receptors which are a cell-specific target of and responsible for the cellular entry of novel coronavirus are localized in lipid rafts, agents that specifically disrupt the relevant rafts may be a drug against coronavirus disease 2019.

scholarly journals Investigation of orf virus structure and morphogenesis using recombinants expressing FLAG-tagged envelope structural proteins: evidence for wrapped virus particles and egress from infected cells

2009 ◽  
Vol 90 (3) ◽  
pp. 614-625 ◽  
Author(s):  
Joanne L. Tan ◽  
Norihito Ueda ◽  
Andrew A. Mercer ◽  
Stephen B. Fleming
Keyword(s):  
Fluorescent Microscopy ◽  
Cellular Membrane ◽  
Immunogold Labelling ◽  
Orf Virus ◽  
Structural Proteins ◽  
Post Translational Modification ◽  
Virus Particles ◽  
Viral Egress ◽  
Infected Cells ◽  
Almost All

Orf virus (ORFV) is the type species of the genus Parapoxvirus, but little is known about the structure or morphogenesis of the virus. In contrast, the structure and morphogenesis of vaccinia virus (VACV) has been extensively studied. VACV has two main infectious forms, mature virion (MV) and extracellular virion (EV). The MV is wrapped by two additional membranes derived from the trans-Golgi to produce a wrapped virion (WV), the outermost of which is lost by cellular membrane fusion during viral egress to form the EV. Genome sequencing of ORFV has revealed that it has homologues of almost all of the VACV structural genes. Notable exceptions are A36R, K2L, A56R and B5R, which are associated with WV and EV envelopes. This study investigated the morphogenesis and structure of ORFV by fusing FLAG peptide to the structural proteins 10 kDa, F1L and ORF-110 to form recombinant viruses. 10 kDa and F1L are homologues of VACV A27L and H3L MV membrane proteins, whilst ORF-110 is homologous to VACV A34R, an EV membrane protein. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG–F1L and FLAG–10 kDa were displayed on the surface of infectious particles, whereas ORF-110–FLAG could not be detected. Western blot analysis of solubilized recombinant ORF-110–FLAG particles revealed that ORF-110–FLAG was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking. Fluorescent microscopy confirmed the prediction that ORF-110–FLAG localized to the Golgi in virus-infected cells. Finally, immunogold labelling of EVs showed that ORF-110–FLAG became exposed on the surface of EV-like particles as a result of egress from the cell.

Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer

1989 ◽  
Vol 9 (1) ◽  
pp. 204-213
Author(s):  
T M Browder ◽  
J S Abrams ◽  
P M Wong ◽  
A W Nienhuis
Keyword(s):  
Signal Sequence ◽  
Polyclonal Antibodies ◽  
Surface Display ◽  
Hematopoietic Cells ◽  
Parental Cell ◽  
Biologically Active ◽  
Parental Cell Line ◽  
Interleukin 3 ◽  
Endogenous Expression ◽  
Autocrine Stimulation

Endogenous expression of the interleukin-3 (IL3) gene introduced with a retrovirus vector renders hematopoietic cells autonomous of exogenous growth factor. To investigate the mechanism of autocrine stimulation, 25 clones were isolated after retrovirus transduction of IL3 into 32D-cl23 or FDC-P1 cells. Medium conditioned by these autonomous IL3-producing clones supported the growth of factor-dependent 32D cells. Although there was a severalfold variation in the amount of IL3 secreted (some clones secreted barely detectable levels), the doubling time of each clone in the absence of added IL3 was identical to that of the parental cell line maximally stimulated by exogenous IL3. Concentrated monoclonal and polyclonal antibodies, both highly effective in neutralizing exogenous IL3, were assayed for ability to inhibit autocrine growth. Minimal inhibitory effects were observed only on washed autocrine clones secreting low levels of IL3. To test the activity of cytoplasmically synthesized IL3, the nucleotides encoding the signal sequence of IL3 were deleted and replaced with an in-frame ATG in the context of a consensus translation initiation sequence. Ten 32D clones expressing this restructured IL3 genome were obtained. Despite the presence of biologically active IL3 in cell lysates, all clones remained dependent on exogenous IL3, with the same dose-response as that found for 32D cells. Our data are most compatible with a mechanism whereby endogenously produced IL3 interacts with its receptor prior to surface display.

Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line

1985 ◽  
Vol 5 (10) ◽  
pp. 2527-2532
Author(s):  
C H Jenh ◽  
P K Geyer ◽  
L F Johnson
Keyword(s):  
Cell Line ◽  
Thymidylate Synthase ◽  
Northern Blot Analysis ◽  
Mouse Cell ◽  
Parental Cell ◽  
S Phase ◽  
Parental Cell Line ◽  
Mouse Cell Line ◽  
Isolated Nuclei ◽  
Mrna Content

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.

Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2973-2979 ◽  
Author(s):  
Anne J. Novak ◽  
Richard J. Bram ◽  
Neil E. Kay ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocyte ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

Quantitative immunoenzymatic assay of human lutropin, with use of a bi-specific monoclonal antibody

1990 ◽  
Vol 36 (1) ◽  
pp. 47-52 ◽  
Author(s):  
G Bugari ◽  
C Poiesi ◽  
A Beretta ◽  
S Ghielmi ◽  
A Albertini
Keyword(s):  
Monoclonal Antibody ◽  
Parental Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parental Cell Line ◽  
Specific Monoclonal Antibody ◽  
Specific Antibodies ◽  
Human Choriogonadotropin ◽  
Enzyme Conjugate ◽  
Capture Antibody ◽  
Immunoenzymatic Assay

Abstract In this immunoenzymatic assay for human lutropin (hLH) we used bi-specific antibodies (BiAbs) obtained from the fusion of two hybridomas producing antibodies to beta-D-galactosidase and hLh. The BiAb complexed with the enzyme beta-D-galactosidase was used as tracer in a double-determinant assay. We compared the assay involving the BiAb (Bi-EIA) with an immunoenzymatic assay (EIA) in which the same capture antibody was used but the tracer was an enzyme-conjugated hLH-specific monoclonal antibody produced by the same parental cell line used to produce the BiAb. The coefficient of correlation (r) between the two assays was 0.979 but the Bi-EIA was more sensitive (detection limits: 0.8 int. units/L for the Bi-EIA, 2.0 int. units/L for the EIA) and more specific (less than 0.04% vs less than 1.2% cross-reactions with human choriogonadotropin). Mean intra- and interassay CVs for the Bi-EIA were 2.9% and 5.9%, respectively. Correlation (r) with an immunoradiometric assay (IRMA, Serono kit) was 0.960, with radioimmunoassay (RIA, Biodata kit) 0.909, and with an enzyme-linked immunosorbent assay (ELISA kit, Specialty Medical Industries Inc.) 0.888, (n = 25). Evidently, bi-specific antibodies can be used successfully in immunoenzymatic assays, and with potentially greater sensitivity and specificity than assay with a traditional antibody-enzyme conjugate.

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