Proteomic Analysis of the Inflamed Intestinal Mucosa Reveals Distinctive Immune Response Profiles in Crohn’s Disease and Ulcerative Colitis

Berndt, Uta; Bartsch, Sebastian; Philipsen, Lars; Danese, Silvio; Wiedenmann, Bertram; Dignaß, Axel; Hämmerle, Marcus; Sturm, Andreas

doi:10.4049/jimmunol.179.1.295

Cited by 38 publications

(43 citation statements)

References 61 publications

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“…We used a MELC library of 31 fluorescence tags composed of antibodies, lectins, and propidium iodide as a nucleic acid dye as recently published (7). The appropriate working dilutions, fluorophore labels, incubation time (15 min), and positions within the MELC run had been established and validated in the course of systematic experiments based on conventional immunohistochemistry and MELC calibration runs (8,9).…”

Section: Melc Technologymentioning

confidence: 99%

“…These CMP motifs are defined as pixelrelated code of 1/0/* ciphering. The ''TopoMiner'' software packages (MelTec GmbH & Co. KG) was used for data mining as described by Schubert et al and Berndt et al (7,9). Three pairwise screens (Wilcoxon rank-sum test, P < 0.01; search depth, n = 5) were performed to detect differences between the three groups (healthy control, ulcerative colitis, and colorectal cancer).…”

Section: Melc Technologymentioning

confidence: 99%

“…Approval of the protocol and consent form was granted by the local ethics committee of the Charité, Berlin, Germany. Biopsies were snap frozen in liquid nitrogen, stored at À80jC, and prepared for MELC analysis as recently described (7).…”

Section: Patients and Sample Preparationmentioning

confidence: 99%

“…This multidimensional microscopic robot technology allows not only high-throughput protein recognition but, furthermore, the combination of localized proteins, providing the opportunity to generate a protein colocalization map. Recently, the identification of disease-relevant protein networks arising from detection of such protein expression patterns in inflammatory skin and bowel disease by using the advantages of the MELC technology has been shown (6,7).…”

Section: Introductionmentioning

confidence: 99%

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Systematic High-Content Proteomic Analysis Reveals Substantial Immunologic Changes in Colorectal Cancer

Berndt

Philipsen²,

Bartsch³

et al. 2008

Cancer Research

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The immune system is a significant determinant of epithelial tumorigenesis, but its role in colorectal cancer pathogenesis is not well understood. The function of the immune system depends upon the integrity of the protein network environment, and thus, we performed MELC immunofluorescence microscopy focusing on the lamina propria. By analyzing structurally intact tissues from colorectal cancer, ulcerative colitis, and healthy colonic mucosa, we used this unique and novel highly multiplexed robotic-imaging technology, which allows visualizing dozens of proteins simultaneously, and explored the toponome in colorectal cancer mucosa for the first time. We identified 1,930 motifs that distinguish control from colorectal cancer tissue. In colorectal cancer, the number of activated T cells is increased, explained by a lack of bax, caspase-3, and caspase-8. Whereas CD4 + CD25 + T cells are decreased and are, other than in ulcerative colitis, not activated, cytotoxic T cells are significantly increased in colorectal cancer. Furthermore, the number of activated human lymphocyte antigen (HLA)-DR + T-cells is increased in colorectal cancer, pointing to an altered antigen presentation. In colorectal cancer, CD3 + CD29 + expression and assembly of the LFA-1 and LFA-3 receptor are differentially changed, indicating a distinct regulation of T-cell adhesion in colorectal cancer. We also identified increased numbers of natural killer and CD44 + cells in the colorectal cancer mucosa and nuclear factor-KB as regulator of apoptosis in these cell populations. High-content proteomic analysis showed that colorectal cancer induces a tremendous modification of protein expression profiles in the lamina propria. Thus, topological proteomic analysis may help to unravel the role of the adaptive immune system in colorectal cancer and aid the development of new antitumor immunotherapy approaches.

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Section: Melc Technologymentioning

confidence: 99%

Section: Melc Technologymentioning

confidence: 99%

Section: Patients and Sample Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Systematic High-Content Proteomic Analysis Reveals Substantial Immunologic Changes in Colorectal Cancer

Berndt

Philipsen²,

Bartsch³

et al. 2008

Cancer Research

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“…This technique, called multi-epitope-ligand cartography (MELC), enables the mapping of protein networks, the localization of different cell types, and the analysis of disease-related changes in the proteome (Schubert et al 2006;Berndt et al 2007;Ruetze et al 2010). Cellular targets are labeled with fluorescent tags; subsequently, images of the fluorescence stainings are taken, and the fluorescence dye is completely bleached (Fig.…”

mentioning

confidence: 99%

Murine Whole-Organ Immune Cell Populations Revealed by Multi-epitope-Ligand Cartography

Eckhardt

Ostalecki

Kuczera

et al. 2012

J Histochem Cytochem.

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Our knowledge of increasing numbers of cell subtype and activation status markers enables more and more subtle distinctions of individual cell subpopulations with specific functions. Fluorescence-activated flow cytometry is the most common technology used routinely to analyze cell suspensions. Because flow cytometry is limited by the availability of discriminable fluorescence dyes, analyses with more than six colors are uncommon. Moreover, the location of an individual cell and its interaction with surrounding cells and matrix is lost in flow cytometry but provides additional important information. A better understanding of the phenotypic composition of tissueinfiltrating leukocytes in context with the cellular environment is essential for immunology. Besides the stages of differentiation and cellular activation, cell-cell and cellmatrix interactions are crucial factors defining the biological activity of individual cells in vitro. Immunohistology is the current approach to evaluate the presence and location of certain markers within organelles or membranes of cells in their physiological environment. But, even more than flow cytometry, immunohistology is limited by the number of distinguishable fluorescence dyes. Therefore, serial sections must be studied, but this approach no longer allows investigation of the same cells.Recently, a new technique of automated multidimensional fluorescence microscopy was published to analyze proteome topology on structurally intact single cells as well as within the complexity of a tissue (Schubert et al. 2006). This technique, called multi-epitope-ligand cartography (MELC), enables the mapping of protein networks, the localization of different cell types, and the analysis of 470140J HCXXX10.1369/0022155412470140Eckha rdt et al.MELC Analysis of Whole Murine Organs 2012© The Author(s) 2010 Reprints and permission: sagepub.com/journalsPermissions. SummaryMulti-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1)

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Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections

et al. 2018

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The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.

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Proteomic Analysis of the Inflamed Intestinal Mucosa Reveals Distinctive Immune Response Profiles in Crohn’s Disease and Ulcerative Colitis

Cited by 38 publications

References 61 publications

Systematic High-Content Proteomic Analysis Reveals Substantial Immunologic Changes in Colorectal Cancer

Systematic High-Content Proteomic Analysis Reveals Substantial Immunologic Changes in Colorectal Cancer

Murine Whole-Organ Immune Cell Populations Revealed by Multi-epitope-Ligand Cartography

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections

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