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  1. Artikel ; Online: Immunofluorescence analysis of human eosinophils.

    Almas, Sarah / Fayad, Nawell / Srivastava, Ojas / Siddique, Mujtaba / Das, Sharanya / Touret, Nicolas / Sun, Xuejun / Lacy, Paige

    Journal of immunological methods

    2024  Band 526, Seite(n) 113619

    Abstract: A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the ... ...

    Abstract A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 μg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.
    Mesh-Begriff(e) Humans ; Eosinophils ; Interleukin-9/metabolism ; Interleukin-13/metabolism ; Cytokines/metabolism ; Fluorescent Antibody Technique ; Saponins/metabolism
    Chemische Substanzen Interleukin-9 ; Interleukin-13 ; Cytokines ; Saponins
    Sprache Englisch
    Erscheinungsdatum 2024-01-23
    Erscheinungsland Netherlands
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 120142-6
    ISSN 1872-7905 ; 0022-1759
    ISSN (online) 1872-7905
    ISSN 0022-1759
    DOI 10.1016/j.jim.2024.113619
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  2. Artikel ; Online: Cytokine trafficking of IL-9 and IL-13 through TfnRc

    Almas, Sarah / Fayad, Nawell / Srivastava, Ojas / Siddique, Mujtaba / Touret, Nicolas / Lacy, Paige

    Journal of leukocyte biology

    2020  Band 109, Heft 4, Seite(n) 753–762

    Abstract: Eosinophils are granulocytes that are elevated in lung mucosa in approximately half of patients with allergic asthma. These highly granulated cells can synthesize and secrete many cytokines, including IL-9 and IL-13. We hypothesized that IL-9 and IL-13 ... ...

    Abstract Eosinophils are granulocytes that are elevated in lung mucosa in approximately half of patients with allergic asthma. These highly granulated cells can synthesize and secrete many cytokines, including IL-9 and IL-13. We hypothesized that IL-9 and IL-13 are found as preformed mediators in crystalloid granules and secreted using distinct trafficking pathways. Human eosinophils were purified from peripheral venous blood, adhered to coverslips, and stimulated with platelet activating factor (PAF). Cells were immunolabeled with antibodies to IL-9 or IL-13 and colocalized with markers for secretory organelles, using CD63 for crystalloid granules and transferrin receptor (TfnRc) for vesicles. Fixed cells were imaged using super-resolution microscopy and quantified by colocalization using Pearson's correlation coefficient. IL-9 immunofluorescence increased in a time-dependent manner to PAF, whereas colocalization of IL-9 and CD63 significantly increased from 0.52 to 0.67 after 5 min PAF. Colocalization of IL-9 with TfnRc significantly increased at 60 min of stimulation with PAF (0.54 at 0 min to 0.60 at 60 min). IL-13 showed lower colocalization with CD63 (0.55) than TfnRc (0.63) in unstimulated cells. Upon PAF stimulation, IL-13 intensity transiently decreased at 5 and 60 min, whereas colocalization of IL-13 with CD63 decreased throughout stimulation to 0.43. While colocalization of IL-13 with TfnRc transiently increased to 0.66 at 5 min PAF, it returned to near baseline levels (0.64) after 15 min PAF. Our results suggest that IL-9 and IL-13 are stored in crystalloid granules as well as endosomal structures, and that IL-9 is primarily trafficked to the cell surface via TfnRc
    Mesh-Begriff(e) Adolescent ; Adult ; Cytoplasmic Granules/metabolism ; Eosinophils/metabolism ; Humans ; Interleukin-9/metabolism ; Platelet Activating Factor/metabolism ; Receptors, Transferrin/metabolism ; Tetraspanin 30/metabolism ; Transport Vesicles/metabolism
    Chemische Substanzen Interleukin-9 ; Platelet Activating Factor ; Receptors, Transferrin ; Tetraspanin 30
    Sprache Englisch
    Erscheinungsdatum 2020-09-10
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 605722-6
    ISSN 1938-3673 ; 0741-5400
    ISSN (online) 1938-3673
    ISSN 0741-5400
    DOI 10.1002/JLB.2MA0820-320RR
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 603: Hefte anzeigen Standort:
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  3. Artikel ; Online: Identification of Human Host Substrates of the SARS-CoV-2 M

    Luo, Shu Y / Moussa, Eman W / Lopez-Orozco, Joaquin / Felix-Lopez, Alberto / Ishida, Ray / Fayad, Nawell / Gomez-Cardona, Erik / Wang, Henry / Wilson, Joyce A / Kumar, Anil / Hobman, Tom C / Julien, Olivier

    ACS infectious diseases

    2023  Band 9, Heft 4, Seite(n) 749–761

    Abstract: The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, ... ...

    Abstract The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, M
    Mesh-Begriff(e) Humans ; SARS-CoV-2/metabolism ; COVID-19 ; Peptide Synthases ; Peptide Hydrolases/metabolism
    Chemische Substanzen subtiligase (EC 6.3.2.-) ; Peptide Synthases (EC 6.3.2.-) ; Peptide Hydrolases (EC 3.4.-)
    Sprache Englisch
    Erscheinungsdatum 2023-04-03
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 2373-8227
    ISSN (online) 2373-8227
    DOI 10.1021/acsinfecdis.2c00458
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  4. Artikel ; Online: The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2.

    Lopez-Orozco, Joaquin / Fayad, Nawell / Khan, Juveriya Qamar / Felix-Lopez, Alberto / Elaish, Mohamed / Rohamare, Megha / Sharma, Maansi / Falzarano, Darryl / Pelletier, Jerry / Wilson, Joyce / Hobman, Tom C / Kumar, Anil

    Journal of molecular biology

    2023  Band 435, Heft 16, Seite(n) 168170

    Abstract: Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then ... ...

    Abstract Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.
    Mesh-Begriff(e) Animals ; Humans ; Argonaute Proteins/genetics ; Argonaute Proteins/metabolism ; COVID-19/metabolism ; COVID-19/virology ; MicroRNAs/genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism
    Chemische Substanzen Argonaute Proteins ; MicroRNAs ; RNA, Double-Stranded ; RNA, Small Interfering ; RNA, Viral ; AGO2 protein, human
    Sprache Englisch
    Erscheinungsdatum 2023-06-03
    Erscheinungsland Netherlands
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2023.168170
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 867: Hefte anzeigen Standort:
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  5. Artikel ; Online: Mayaro Virus Non-Structural Protein 2 Circumvents the Induction of Interferon in Part by Depleting Host Transcription Initiation Factor IIE Subunit 2.

    Ishida, Ray / Cole, Jamie / Lopez-Orozco, Joaquin / Fayad, Nawell / Felix-Lopez, Alberto / Elaish, Mohamed / Luo, Shu Yue / Julien, Olivier / Kumar, Anil / Hobman, Tom C

    Cells

    2021  Band 10, Heft 12

    Abstract: Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the ... ...

    Abstract Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus
    Mesh-Begriff(e) Alphavirus/metabolism ; Animals ; Cell Line ; Cell Nucleus/metabolism ; Down-Regulation ; Host-Pathogen Interactions ; Humans ; Interferon Regulatory Factor-3/metabolism ; Interferons/metabolism ; Protein Binding ; Protein Subunits/metabolism ; Protein Transport ; RNA Polymerase II/metabolism ; Transcription Factors, TFII/metabolism ; Transcription, Genetic ; Viral Nonstructural Proteins/metabolism
    Chemische Substanzen IRF3 protein, human ; Interferon Regulatory Factor-3 ; Protein Subunits ; Transcription Factors, TFII ; Viral Nonstructural Proteins ; transcription factor TFIIE ; Interferons (9008-11-1) ; RNA Polymerase II (EC 2.7.7.-)
    Sprache Englisch
    Erscheinungsdatum 2021-12-12
    Erscheinungsland Switzerland
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2661518-6
    ISSN 2073-4409 ; 2073-4409
    ISSN (online) 2073-4409
    ISSN 2073-4409
    DOI 10.3390/cells10123510
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  6. Artikel ; Online: SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors.

    Kumar, Anil / Ishida, Ray / Strilets, Tania / Cole, Jamie / Lopez-Orozco, Joaquin / Fayad, Nawell / Felix-Lopez, Alberto / Elaish, Mohamed / Evseev, Danyel / Magor, Katharine E / Mahal, Lara K / Nagata, Les P / Evans, David H / Hobman, Tom C

    Journal of virology

    2021  Band 95, Heft 13, Seite(n) e0026621

    Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) ... ...

    Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection.
    Mesh-Begriff(e) Animals ; COVID-19/immunology ; COVID-19/virology ; Chlorocebus aethiops ; Coronavirus Nucleocapsid Proteins/metabolism ; HEK293 Cells ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3/metabolism ; Interferon Type I/antagonists & inhibitors ; Interferon Type I/metabolism ; Phosphoproteins/metabolism ; SARS-CoV-2/metabolism ; SARS-CoV-2/pathogenicity ; STAT2 Transcription Factor/metabolism ; Signal Transduction ; TYK2 Kinase/metabolism ; Vero Cells ; Viral Nonstructural Proteins/metabolism
    Chemische Substanzen Coronavirus Nucleocapsid Proteins ; Interferon Regulatory Factor-3 ; Interferon Type I ; NSP1 protein, SARS-CoV-2 ; Phosphoproteins ; STAT2 Transcription Factor ; Viral Nonstructural Proteins ; nucleocapsid phosphoprotein, SARS-CoV-2 ; TYK2 Kinase (EC 2.7.10.2)
    Sprache Englisch
    Erscheinungsdatum 2021-06-10
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80174-4
    ISSN 1098-5514 ; 0022-538X
    ISSN (online) 1098-5514
    ISSN 0022-538X
    DOI 10.1128/JVI.00266-21
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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