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Article ; Online: Imaging Mass Cytometry for In Situ Immune Profiling.

Hu, Kevin / Harman, Andrew / Baharlou, Heeva

Methods in molecular biology (Clifton, N.J.)

2024 Volume 2779, Page(s) 407–423

Abstract: The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations ... ...

Abstract	The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.
MeSH term(s)	RNA ; Staining and Labeling ; Proteins ; Image Cytometry/methods ; Flow Cytometry/methods
Chemical Substances	RNA (63231-63-0) ; Proteins
Language	English
Publishing date	2024-03-25
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3738-8_19
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Article ; Online: spicyR: spatial analysis of in situ cytometry data in R.

Canete, Nicolas P / Iyengar, Sourish S / Ormerod, John T / Baharlou, Heeva / Harman, Andrew N / Patrick, Ellis

Bioinformatics (Oxford, England)

2022 Volume 38, Issue 11, Page(s) 3099–3105

Abstract: Motivation: High parameter histological techniques have allowed for the identification of a variety of distinct cell types within an image, providing a comprehensive overview of the tissue environment. This allows the complex cellular architecture and ... ...

Abstract	Motivation: High parameter histological techniques have allowed for the identification of a variety of distinct cell types within an image, providing a comprehensive overview of the tissue environment. This allows the complex cellular architecture and environment of diseased tissue to be explored. While spatial analysis techniques have revealed how cell-cell interactions are important within the disease pathology, there remains a gap in exploring changes in these interactions within the disease process. Specifically, there are currently few established methods for performing inference on cell-type co-localization changes across images, hindering an understanding of how cellular environments change with a disease pathology. Results: We have developed the spicyR R package to perform inference on changes in the spatial co-localization of types across groups of images. Application to simulated data demonstrates a high sensitivity and specificity. We the utility of spicyR by applying it to a type 1 diabetes imaging mass cytometry dataset, revealing changes in cellular associations that were relevant to the disease progression. Ultimately, spicyR allows changes in cellular environments to be explored under different pathologies or disease states. Availability and implementation: R package is freely available at http://bioconductor.org/packages/release/bioc/html/spicyR.html and shiny app implementation at http://shiny.maths.usyd.edu.au/spicyR/. Supplementary information: Supplementary data are available at Bioinformatics online.
MeSH term(s)	Software ; Spatial Analysis
Language	English
Publishing date	2022-04-14
Publishing country	England
Document type	Review ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btac268
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Article ; Online: Defining the landscape of human epidermal mononuclear phagocytes.

Bertram, Kirstie M / O'Neil, Thomas R / Vine, Erica E / Baharlou, Heeva / Cunningham, Anthony L / Harman, Andrew N

Immunity

2023 Volume 56, Issue 3, Page(s) 459–460

MeSH term(s)	Humans ; Dendritic Cells ; Epidermis ; Skin ; Langerhans Cells ; Phagocytes
Language	English
Publishing date	2023-04-05
Publishing country	United States
Document type	Letter ; Comment
ZDB-ID	1217235-2
ISSN	1097-4180 ; 1074-7613
ISSN (online)	1097-4180
ISSN	1074-7613
DOI	10.1016/j.immuni.2023.02.001
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Zs.A 4227: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Mass Cytometry Imaging for the Study of Human Diseases-Applications and Data Analysis Strategies.

Baharlou, Heeva / Canete, Nicolas P / Cunningham, Anthony L / Harman, Andrew N / Patrick, Ellis

Frontiers in immunology

2019 Volume 10, Page(s) 2657

Abstract: High parameter imaging is an important tool in the life sciences for both discovery and healthcare applications. Imaging Mass Cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI) are two relatively recent technologies which enable clinical samples to ... ...

Abstract	High parameter imaging is an important tool in the life sciences for both discovery and healthcare applications. Imaging Mass Cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI) are two relatively recent technologies which enable clinical samples to be simultaneously analyzed for up to 40 parameters at subcellular resolution. Importantly, these "Mass Cytometry Imaging" (MCI) modalities are being rapidly adopted for studies of the immune system in both health and disease. In this review we discuss, first, the various applications of MCI to date. Second, due to the inherent challenge of analyzing high parameter spatial data, we discuss the various approaches that have been employed for the processing and analysis of data from MCI experiments.
MeSH term(s)	Data Analysis ; Flow Cytometry/methods ; Humans ; Image Cytometry/methods
Language	English
Publishing date	2019-11-14
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2019.02657
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Article ; Online: AFid: a tool for automated identification and exclusion of autofluorescent objects from microscopy images.

Baharlou, Heeva / Canete, Nicolas P / Bertram, Kirstie M / Sandgren, Kerrie J / Cunningham, Anthony L / Harman, Andrew N / Patrick, Ellis

Bioinformatics (Oxford, England)

2020 Volume 37, Issue 4, Page(s) 559–567

Abstract: Motivation: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical ... ...

Abstract	Motivation: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical treatment of sections or specialized instrumentation and software to 'unmix' autofluorescent signals. Importantly, these approaches are pre-emptive and there are currently no methods to deal with autofluorescence in acquired fluorescence microscopy images. Results: To address this, we developed Autofluorescence Identifier (AFid). AFid identifies autofluorescent pixels as discrete objects in multi-channel images post-acquisition. These objects can then be tagged for exclusion from downstream analysis. We validated AFid using images of FFPE human colorectal tissue stained for common immune markers. Further, we demonstrate its utility for image analysis where its implementation allows the accurate measurement of HIV-Dendritic cell interactions in a colorectal explant model of HIV transmission. Therefore, AFid represents a major leap forward in the extraction of useful data from images plagued by autofluorescence by offering an approach that is easily incorporated into existing workflows and that can be used with various samples, staining panels and image acquisition methods. We have implemented AFid in ImageJ, Matlab and R to accommodate the diverse image analysis community. Availability and implementation: AFid software is available at https://ellispatrick.github.io/AFid. Supplementary information: Supplementary data are available at Bioinformatics online.
MeSH term(s)	Histological Techniques ; Humans ; Image Processing, Computer-Assisted ; Microscopy, Fluorescence ; Software ; Workflow
Language	English
Publishing date	2020-10-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btaa780
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Article ; Online: Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Tong, Orion / Duette, Gabriel / O'Neil, Thomas R / Royle, Caroline M / Rana, Hafsa / Johnson, Blake / Popovic, Nicole / Dervish, Suat / Brouwer, Michelle A E / Baharlou, Heeva / Patrick, Ellis / Ctercteko, Grahame / Palmer, Sarah / Lee, Eunok / Hunter, Eric / Harman, Andrew N / Cunningham, Anthony L / Nasr, Najla

PLoS pathogens

2021 Volume 17, Issue 4, Page(s) e1009522

Abstract: Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the ... ...

Abstract	Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.
MeSH term(s)	CD4-Positive T-Lymphocytes/immunology ; CD4-Positive T-Lymphocytes/virology ; Dendritic Cells/immunology ; Dendritic Cells/virology ; Flow Cytometry ; HIV/genetics ; HIV/immunology ; HIV/physiology ; HIV Core Protein p24/genetics ; HIV Core Protein p24/metabolism ; HIV Infections/immunology ; HIV Infections/virology ; Humans ; Interferon-alpha/metabolism ; Myeloid Cells/immunology ; Myeloid Cells/virology ; Phenotype
Chemical Substances	HIV Core Protein p24 ; Interferon-alpha
Language	English
Publishing date	2021-04-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1009522
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Article ; Online: Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

Doyle, Chloe M / Vine, Erica E / Bertram, Kirstie M / Baharlou, Heeva / Rhodes, Jake W / Dervish, Suat / Gosselink, Martijn P / Di Re, Angelina / Collins, Geoffrey P / Reza, Faizur / Toh, James W T / Pathma-Nathan, Nimalan / Ahlenstiel, Golo / Ctercteko, Grahame / Cunningham, Anthony L / Harman, Andrew N / Byrne, Scott N

Frontiers in immunology

2021 Volume 12, Page(s) 727952

Abstract: The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ... ...

Abstract	The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin
MeSH term(s)	Biomarkers/metabolism ; Cell Separation ; Cells, Cultured ; Colon/cytology ; Cytokines/metabolism ; Dendritic Cells/immunology ; Dendritic Cells/metabolism ; Flow Cytometry ; Humans ; Ileum/cytology ; Intestinal Mucosa/cytology ; Jejunum/cytology ; Macrophages/immunology ; Macrophages/metabolism ; Monocytes/immunology ; Monocytes/metabolism ; Phagocytes/immunology ; Phagocytes/metabolism ; Phenotype
Chemical Substances	Biomarkers ; Cytokines
Language	English
Publishing date	2021-09-01
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.727952
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Article ; Online: An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

Cell reports

2022 Volume 40, Issue 12, Page(s) 111385

Abstract: The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we ... ...

Abstract	The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4
MeSH term(s)	CD4-Positive T-Lymphocytes ; Colorectal Neoplasms/pathology ; Dendritic Cells ; HIV Infections ; HIV-1 ; Host-Pathogen Interactions ; Humans
Language	English
Publishing date	2022-09-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2022.111385
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Article ; Online: Langerhans cells and sexual transmission of HIV and HSV.

Botting, Rachel A / Rana, Hafsa / Bertram, Kirstie M / Rhodes, Jake W / Baharlou, Heeva / Nasr, Najla / Cunningham, Anthony L / Harman, Andrew N

Reviews in medical virology

2017 Volume 27, Issue 2

Abstract: Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role ... ...

Abstract	Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role in the host mounting an appropriate immune response. As such, viruses have evolved complex strategies to manipulate these cells to facilitate successful transmission. One of best studied examples is HIV, which manipulates the natural function of these cells to interact with CD4 T cells, which are the main target cell for HIV in which rapid replication occurs. However, there is controversy in the literature as to the role that LCs play in this process. Langerhans cells also play a key role in the way the body mounts an immune response to HSV, and there is also a complex interplay between the transmission of HSV and HIV that involves LCs. In this article, we review both past and present literatures with a particular focus on a few very recent studies that shed new light on the role that LCs play in the transmission and immune response to these 2 pathogens.
MeSH term(s)	HIV Infections/transmission ; Herpes Genitalis/transmission ; Host-Pathogen Interactions ; Humans ; Langerhans Cells/immunology ; Langerhans Cells/virology
Language	English
Publishing date	2017-03
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1086043-5
ISSN	1099-1654 ; 1052-9276
ISSN (online)	1099-1654
ISSN	1052-9276
DOI	10.1002/rmv.1923
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Article ; Online: Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Rhodes, Jake W / Botting, Rachel A / Bertram, Kirstie M / Vine, Erica E / Rana, Hafsa / Baharlou, Heeva / Vegh, Peter / O'Neil, Thomas R / Ashhurst, Anneliese S / Fletcher, James / Parnell, Grant P / Graham, J Dinny / Nasr, Najla / Lim, Jake J K / Barnouti, Laith / Haertsch, Peter / Gosselink, Martijn P / Di Re, Angelina / Reza, Faizur /

Ctercteko, Grahame / Jenkins, Gregory J / Brooks, Andrew J / Patrick, Ellis / Byrne, Scott N / Hunter, Eric / Haniffa, Muzlifah A / Cunningham, Anthony L / Harman, Andrew N

Nature communications

2021 Volume 12, Issue 1, Page(s) 2147

Abstract: Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal ... ...

Abstract	Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14
MeSH term(s)	Anal Canal/cytology ; Antigens, CD/metabolism ; CD4-Positive T-Lymphocytes/immunology ; Cell Shape ; Collagenases/metabolism ; Dendritic Cells/metabolism ; Dermis/metabolism ; Genitalia/cytology ; HIV Infections/immunology ; HIV Infections/virology ; HIV-1/physiology ; Humans ; Lectins, C-Type/metabolism ; Lipopolysaccharide Receptors/metabolism ; Mannose-Binding Lectins/metabolism ; Monocytes/metabolism ; Mucous Membrane/metabolism ; Phagocytes/metabolism ; Phenotype ; Receptors, CCR5/metabolism ; Sialic Acid Binding Ig-like Lectin 1/metabolism ; Transcription, Genetic
Chemical Substances	Antigens, CD ; CCR5 protein, human ; CD207 protein, human ; Lectins, C-Type ; Lipopolysaccharide Receptors ; Mannose-Binding Lectins ; Receptors, CCR5 ; Sialic Acid Binding Ig-like Lectin 1 ; Collagenases (EC 3.4.24.-)
Language	English
Publishing date	2021-04-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-22375-x
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