Detection by 4-parameter microscopic imaging and increase of rare mononuclear blood leukocyte types expressing the FcγRIII receptor (CD 16) for immunoglobulin G in human sporadic amyotrophic lateral sclerosis (ALS)

Schubert, Walter; Schwan, Horst

doi:10.1016/0304-3940(95)11956-w

Cited by 11 publications

(11 citation statements)

References 19 publications

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“…Five-micrometer thick cryosections were prepared according to earlier protocols (24): fixed in ice-cold acetone at 220°C for 20 min, air-dried, and then rehydrated in PBS (pH 7.4) at room temperature. Peripheral blood mononuclear cells (PBMC) were prepared as described (25). Briefly, PBMC were isolated from the blood of a healthy volunteer by a Ficoll isolation procedure, placed on an object slide, air-dried, and then fixed in ice-cold acetone and rehydrated at room temperature as described earlier.…”

Section: Preparation Of Cells and Tissue Sectionsmentioning

confidence: 99%

“…First, peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Briefly, PBMC isolated from the blood and fixed on cover slips by routine procedures (25) were subjected to the MELK procedure. In the present example, altogether nine cycles with 2 mAbs per cycle were performed showing highly heterogeneous cell surface protein-epitope distribution patterns (Fig.…”

Section: Mapping Mononuclear Leukocytes In Multiple Immune Compartmentsmentioning

confidence: 99%

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A three‐symbol code for organized proteomes based on cyclical imaging of protein locations

Schubert

2007

Cytometry Pt A

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Background: A major challenge in the post genomic era is to map and decipher the functional molecular networks of proteins directly in a cell or a tissue. This task requires technologies for the colocalization of random numbers of different molecular components (e.g. proteins) in one sample in one experiment. Methods: Multi‐epitope‐ligand‐“kartographie” (MELK) was developed as a microscopic imaging technology running cycles of iterative fluorescence tagging, imaging, and bleaching, to colocalize a large number of proteins in one sample (morphologically intact routinely fixed cells or tissue). Results: In the present study, 18 different cell surface proteins were colocalized by MELK in cells and tissue sections in different compartments of the human immune system. From the resulting sets of multidimensional binary vectors the most prominent groups of protein–epitope arrangements were extracted and imaged as protein “toponome” maps providing direct insight in the higher order topological organization of immune compartments uncovering new tissue domains. The data sets suggest that protein networks, topologically organized in proteomes in situ, obey a unique protein‐colocation and ‐anticolocation code describable by three symbols. Conclusion: The technology has the potential to colocalize hundreds of proteins and other molecular components in one sample and may offer many applications in biology and medicine. © 2007 International Society for Analytical Cytology.

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Section: Preparation Of Cells and Tissue Sectionsmentioning

confidence: 99%

Section: Mapping Mononuclear Leukocytes In Multiple Immune Compartmentsmentioning

confidence: 99%

A three‐symbol code for organized proteomes based on cyclical imaging of protein locations

Schubert

2007

Cytometry Pt A

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“…Sections were treated as mentioned above. Serial incubation with the following primary conjugated antibodies (CD4-PE and CD8-FITC, CD3-PE and TCR gd-FITC, CD56-PE and CD45RO-FITC, CD64-PE and CD103-FITC, and CD18-PE) was performed as described [15,19]. In short, immunofluorescent sections were examined with a Zeiss microscope equipped with a 50-W mercury lamp and a 40 × water immersion objective (Zeiss, Jena, Germany).…”

Section: Multi-epitope Imagingmentioning

confidence: 99%

Effector T lymphocyte subsets in human pancreatic cancer: detection of CD8+ CD18+ cells and CD8+ CD103+ cells by multi-epitope imaging

Ademmer¹,

Ebert²,

Müller-Ostermeyer

et al. 1998

Clinical and Experimental Immunology

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Pancreatic cancer is characterized by an increasing incidence and an extremely poor prognosis. It is resistant to most of the conventional treatment modalities. Histomorphologically, it presents with a strong desmoplastic reaction around cancer cells, and lymphocytes are typically localized as aggregates in the fibrotic interstitial tissue. Using the method of multi-epitope imaging with fluorochrome-tagged specific MoAbs which allows the simultaneous localization and characterization of T cells in tissues, we studied phenotypes and distribution of tumour-infiltrating lymphocytes (TIL) in pancreatic cancer. CD3+ T cells comprised up to 90% of the tumour-infiltrating cells which were either CD4+ or CD8+, most of them being memory cells (CD45RO+). In decreasing order of frequency, T lymphocytes carried the markers for CD45RO, CD18, CD103 and TCR gammadelta. Very few natural killer cells (CD56+) were observed. Twenty percent of CD8+ were labelled with CD103. These CD8+CD103+ T cells, analogous to the gut intraepithelial lymphocytes (IEL), were found in the fibrous interstitial tissue. Furthermore, an inverse correlation was found between the expression of CD18, the beta2-integrin, which mediates adhesion of activated lymphocytes, and CD45RO in the CD8+ subset of TIL (P = 0.046). In conclusion, phenotyping of T lymphocytes in pancreatic cancer raises the possibility that pancreatic cancer cells develop several strategies to escape the T cell-induced cytolysis by (i) the aggregation of cytotoxic CD8+CD103+ T cells in the fibrous tissue distant from the tumour cells, and (ii) the presence of CD18-bearing cells which lack the expression of the activation marker CD45RO.

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“…I interpreted this finding likely to reflect a substantial intrinsic alteration of the cellular immune system rather than merely a reaction to a neurodegeneration, and I took a closer look at the immune cell differentiation. I initiated a first quantitative four-parameter screening study 63 . We found highly unusual if not abnormal immune cell subsets in a number of ALS patients expressing CD16+ CD8+ and CD57+ or CD16+ CD8+ CD57− cell surface clusters only in sporadic ALS with supranuclear palsy and in sporadic ALS with predominant involvement of the upper motor neuron.…”

Section: Icm-b Ased a Pproach Tmentioning

confidence: 99%

Advances in toponomics drug discovery: Imaging cycler microscopy correctly predicts a therapy method of amyotrophic lateral sclerosis

Schubert

2015

Cytometry Pt A

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An imaging cycler microscope (ICM) is a fully automated (epi)fluorescence microscope which overcomes the spectral resolution limit resulting in parameter-and dimensionunlimited fluorescence imaging. This enables the spatial resolution of large molecular systems with their emergent topological properties (toponome) in morphologically intact cells and tissues displaying thousands of multi protein assemblies at a time. The resulting combinatorial geometry of these systems has been shown to be key for invivo/in-situ detection of lead proteins controlling protein network topology and (dys)-function: If lead proteins are blocked or downregulated the corresponding disease protein network disassembles. Here, correct therapeutic predictions are exemplified for ALS. ICM drug target studies have discovered an 18-dimensional cell surface molecular system in ALS-PBMC with a lead drug target protein, whose therapeutic downregulation is now reported to show statistically significant effect with stop of disease progression in one third of the ALS patients. Together, this clinical and the earlier experimental validations of the ICM approach indicate that ICM readily discovers in vivo robustness nodes of disease with lead proteins controlling them. Breaking in vivo robustness nodes using drugs against their lead proteins is likely to overcome current high drug attrition rates. V C 2015 The Author. Published by Wiley Periodicals, Inc, on behalf of ISAC.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Detection by 4-parameter microscopic imaging and increase of rare mononuclear blood leukocyte types expressing the FcγRIII receptor (CD 16) for immunoglobulin G in human sporadic amyotrophic lateral sclerosis (ALS)

Cited by 11 publications

References 19 publications

A three‐symbol code for organized proteomes based on cyclical imaging of protein locations

A three‐symbol code for organized proteomes based on cyclical imaging of protein locations

Effector T lymphocyte subsets in human pancreatic cancer: detection of CD8+ CD18+ cells and CD8+ CD103+ cells by multi-epitope imaging

Advances in toponomics drug discovery: Imaging cycler microscopy correctly predicts a therapy method of amyotrophic lateral sclerosis

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