scholarly journals Modeling Adenovirus Latency in Human Lymphocyte Cell Lines

2010 ◽  
Vol 84 (17) ◽  
pp. 8799-8810 ◽  
Author(s):  
Yange Zhang ◽  
Wen Huang ◽  
David A. Ornelles ◽  
Linda R. Gooding
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Virus Genome ◽  
Mrna Levels ◽  
Late Gene Expression ◽  
Retroviral Transduction ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Infected Cells ◽  
Adenovirus Receptor

ABSTRACT Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection.

scholarly journals CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV

2010 ◽  
Vol 84 (22) ◽  
pp. 11614-11623 ◽  
Author(s):  
Shiho Miura ◽  
Kei Kawana ◽  
Danny J. Schust ◽  
Tomoyuki Fujii ◽  
Terufumi Yokoyama ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cell Surface ◽  
Cell Lines ◽  
Cancer Cell Line ◽  
Interleukin 12 ◽  
Cervical Cancer Cell Line ◽  
Mrna Levels ◽  
Infected Cells

ABSTRACT CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

scholarly journals Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

2018 ◽  
Vol 115 (19) ◽  
pp. E4473-E4482 ◽  
Author(s):  
John K. Lee ◽  
Nathanael J. Bangayan ◽  
Timothy Chai ◽  
Bryan A. Smith ◽  
Tiffany E. Pariva ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Surface ◽  
Cell Lines ◽  
Treatment Resistance ◽  
Specific Cell ◽  
Surface Markers ◽  
Mrna Levels ◽  
Cell Surface Markers ◽  
Cell Surface Antigens ◽  
Cancer Subtypes

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

scholarly journals Cyclopropene derivatives of aminosugars for metabolic glycoengineering

2019 ◽  
Vol 15 ◽  
pp. 584-601 ◽  
Author(s):  
Jessica Hassenrück ◽  
Valentin Wittmann
Keyword(s):  
Cell Surface ◽  
Diels Alder ◽  
Inverse Electron Demand ◽  
Distinct Cell ◽  
Chemical Reporter ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Metabolic Glycoengineering ◽  
Derivatives Of ◽  
Electron Demand

Cyclopropenes have been proven valuable chemical reporter groups for metabolic glycoengineering (MGE). They readily react with tetrazines in an inverse electron-demand Diels–Alder (DAinv) reaction, a prime example of a bioorthogonal ligation reaction, allowing their visualization in biological systems. Here, we present a comparative study of six cyclopropene-modified hexosamine derivatives and their suitability for MGE. Three mannosamine derivatives in which the cyclopropene moiety is attached to the sugar by either an amide or a carbamate linkage and that differ by the presence or absence of a stabilizing methyl group at the double bond have been examined. We determined their DAinv reaction kinetics and their labeling intensities after metabolic incorporation. To determine the efficiencies by which the derivatives are metabolized to sialic acids, we synthesized and investigated the corresponding cyclopropane derivatives because cyclopropenes are not stable under the analysis conditions. From these experiments, it became obvious that N-(cycloprop-2-en-1-ylcarbonyl)-modified (Cp-modified) mannosamine has the highest metabolic acceptance. However, carbamate-linked N-(2-methylcycloprop-2-en-1-ylmethyloxycarbonyl)-modified (Cyoc-modified) mannosamine despite its lower metabolic acceptance results in the same cell-surface labeling intensity due to its superior reactivity in the DAinv reaction. Based on the high incorporation efficiency of the Cp derivative we synthesized and investigated two new Cp-modified glucosamine and galactosamine derivatives. Both compounds lead to comparable, distinct cell-surface staining after MGE. We further found that the amide-linked Cp-modified glucosamine derivative but not the Cyoc-modified glucosamine is metabolically converted to the corresponding sialic acid.

Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens

2014 ◽  
pp. n/a-n/a ◽  
Author(s):  
Martina De Vita ◽  
Valentina Catzola ◽  
Alexia Buzzonetti ◽  
Marco Fossati ◽  
Alessandra Battaglia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Surface Staining ◽  
Surface Staining

scholarly journals SARS-COV2 Envelope Protein (E) interacts with the Lysophosphatidic Acid Receptor 1 (LPAR1) from humans

2020 ◽  
Author(s):  
GIRISH NALLUR
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Protein Interactions ◽  
Biological Significance ◽  
Cell Surface Receptor ◽  
Proteomic Screen ◽  
E Protein ◽  
Cell Surface Staining ◽  
Surface Receptor ◽  
Strong Candidate

Abstract A proteomic screen of human proteins interacting with the SARS-COV2 Envelope (E) protein identified LPAR1 as a strong candidate. Physical association of E protein and LPAR1 was confirmed by co-immunoprecipitation and cell surface staining. LPAR1-E protein interaction was confirmed in all eight human cell lines tested. Many additional proteins participating in the E protein interactions network were also enriched from each of the cell lines, some of which were cell type specific. These findings suggest that LPAR1 is likely a cell surface receptor for the E protein, and pave the way for follow-on studies aimed at understanding the biological significance of the interactions in SARS-COV disease, including the signaling mechanisms.

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