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Article ; Online: Application of Digital Holographic Microscopy to Analyze Changes in T-Cell Morphology in Response to Bacterial Challenge

Kari Lavinia vom Werth / Björn Kemper / Stefanie Kampmeier / Alexander Mellmann

Cells, Vol 12, Iss 762, p

2023 Volume 762

Abstract: Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific ... ...

Abstract	Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific morphological changes in human primary T-cells exposed to various bacterial species and strains. Cells were challenged with sterile bacterial determinants, i.e., membrane vesicles or culture supernatants, derived from different Gram-positive and Gram-negative bacteria. Timelapse QPI by digital holographic microscopy (DHM) was applied to capture changes in T-cell morphology over time. After numerical reconstruction and image segmentation, we calculated single cell area, circularity and mean phase contrast. Upon bacterial challenge, T-cells underwent rapid morphological changes such as cell shrinkage, alterations of mean phase contrast and loss of cell integrity. Time course and intensity of this response varied between both different species and strains. The strongest effect was observed for treatment with S. aureus -derived culture supernatants that led to complete lysis of the cells. Furthermore, cell shrinkage and loss of circular shape was stronger in Gram-negative than in Gram-positive bacteria. Additionally, T-cell response to bacterial virulence factors was concentration-dependent, as decreases in cellular area and circularity were enhanced with increasing concentrations of bacterial determinants. Our findings clearly indicate that T-cell response to bacterial stress depends on the causative pathogen, and specific morphological alterations can be detected using DHM.
Keywords	digital holographic microscopy ; sepsis ; bacteria ; T-cells ; cell morphology ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2023-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells

Ralph Rose / Björn Kemper / Albrecht Schwab / Eberhard Schlatter / Bayram Edemir

Scientific Reports, Vol 11, Iss 1, Pp 1-

2021 Volume 12

Abstract: Abstract Aquaporin-2–4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this ... ...

Abstract	Abstract Aquaporin-2–4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2–4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2–3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Investigating Morphological Changes of T-lymphocytes after Exposure with Bacterial Determinants for Early Detection of Septic Conditions

Kari Lavinia vom Werth / Theresa Wörmann / Björn Kemper / Philipp Kümpers / Stefanie Kampmeier / Alexander Mellmann

Microorganisms, Vol 10, Iss 391, p

2022 Volume 391

Abstract: Sepsis is a leading cause of morbidity and mortality, annually affecting millions of people worldwide. Immediate treatment initiation is crucial to improve the outcome but despite great progress, early identification of septic patients remains a ... ...

Abstract	Sepsis is a leading cause of morbidity and mortality, annually affecting millions of people worldwide. Immediate treatment initiation is crucial to improve the outcome but despite great progress, early identification of septic patients remains a challenge. Recently, white blood cell morphology was proposed as a new biomarker for sepsis diagnosis. In this proof-of-concept study, we aimed to investigate the effect of different bacteria and their determinants on T-lymphocytes by digital holographic microscopy (DHM). We hypothesize that species- and strain-specific morphological changes occur, which may offer a new approach for early sepsis diagnosis and identification of the causative agent. Jurkat cells as a model system were exposed to different S. aureus or E. coli strains either using sterile determinants or living bacteria. Time-lapse DHM was applied to analyze cellular morphological changes. There were not only living bacteria but also membrane vesicles and sterile culture supernatant-induced changes of cell area, circularity, and mean phase contrast. Interestingly, different cellular responses occurred depending on both the species and strain of the causative bacteria. Our findings suggest that investigation of T-lymphocyte morphology might provide a promising tool for the early identification of bacterial infections and possibly discrimination between different causative agents. Distinguishing gram-positive from gram-negative infection would already offer a great benefit for the proper administration of antibiotics.
Keywords	sepsis ; T-lymphocyte ; cell morphology ; digital holographic microscopy ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Quantitative Phase Imaging Using Digital Holographic Microscopy to Assess the Degree of Intestinal Inflammation in Patients with Ulcerative Colitis

Arne Bokemeyer / Joost Buskermolen / Steffi Ketelhut / Phil-Robin Tepasse / Richard Vollenberg / Jonel Trebicka / Hartmut H. Schmidt / Michael Vieth / Dominik Bettenworth / Björn Kemper

Journal of Clinical Medicine, Vol 12, Iss 4067, p

2023 Volume 4067

Abstract: Ulcerative colitis (UC) is characterized by chronic inflammation of the colorectum. Histological remission has emerged as a potential future treatment goal; however, the histopathological assessment of intestinal inflammation in UC remains challenging ... ...

Abstract	Ulcerative colitis (UC) is characterized by chronic inflammation of the colorectum. Histological remission has emerged as a potential future treatment goal; however, the histopathological assessment of intestinal inflammation in UC remains challenging with a multitude of available scoring systems and the need for a pathologist with expertise in inflammatory bowel disease (IBD). In previous studies, quantitative phase imaging (QPI) including digital holographic microscopy (DHM) was successfully applied as an objective method for stain-free quantification of the degree of inflammation in tissue sections. Here, we evaluated the application of DHM for the quantitative assessment of histopathological inflammation in patients with UC. In our study, endoscopically obtained colonic and rectal mucosal biopsy samples from 21 patients with UC were analyzed by capturing DHM-based QPI images that were subsequently evaluated using the subepithelial refractive index (RI). The retrieved RI data were correlated with established histological scoring systems including the Nancy index (NI) as well as with endoscopic and clinical findings. As a primary endpoint, we found a significant correlation between the DHM-based retrieved RI and the NI (R 2 = 0.251, p < 0.001). Furthermore, RI values correlated with the Mayo endoscopic subscore (MES; R 2 = 0.176, p < 0.001). An area under the receiver operating characteristics (ROC) curve of 0.820 confirms the subepithelial RI as a reliable parameter to distinguish biopsies with histologically active UC from biopsies without evidence of active disease as determined by conventional histopathological examination. An RI higher than 1.3488 was found to be the most sensitive and specific cut-off value to identify histologically active UC (sensitivity of 84% and specificity of 72%). In conclusion, our data demonstrate DHM to be a reliable tool for the quantitative assessment of mucosal inflammation in patients with UC.
Keywords	quantitative phase imaging ; digital holographic microscopy ; inflammatory bowel disease ; ulcerative colitis ; inflammation ; refractive index ; Medicine ; R
Subject code	610
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The Influence of Capsaicin on the Integrity of Microvascular Endothelial Cell Monolayers

Mathias Kaiser / Malgorzata Burek / Stefan Britz / Frauke Lankamp / Steffi Ketelhut / Björn Kemper / Carola Förster / Christian Gorzelanny / Francisco M. Goycoolea

International Journal of Molecular Sciences, Vol 20, Iss 1, p

2018 Volume 122

Abstract: Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB ... ...

Abstract	Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.
Keywords	tight junctions ; capsaicin ; endothelial cells ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2018-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Knockdown of Musashi RNA Binding Proteins Decreases Radioresistance but Enhances Cell Motility and Invasion in Triple-Negative Breast Cancer

Fabian M. Troschel / Annemarie Minte / Yahia Mahmoud Ismail / Amr Kamal / Mahmoud Salah Abdullah / Sarah Hamdy Ahmed / Marie Deffner / Björn Kemper / Ludwig Kiesel / Hans Theodor Eich / Sherif Abdelaziz Ibrahim / Martin Götte / Burkhard Greve

International Journal of Molecular Sciences, Vol 21, Iss 6, p

2020 Volume 2169

Abstract: The therapeutic potential of Musashi (MSI) RNA-binding proteins, important stemness-associated gene expression regulators, remains insufficiently understood in breast cancer. This study identifies the interplay between MSI protein expression, stem cell ... ...

Abstract	The therapeutic potential of Musashi (MSI) RNA-binding proteins, important stemness-associated gene expression regulators, remains insufficiently understood in breast cancer. This study identifies the interplay between MSI protein expression, stem cell characteristics, radioresistance, cell invasiveness and migration. MSI-1, MSI-2 and Notch pathway elements were investigated via quantitative polymerase chain reaction (qPCR) in 19 triple-negative breast cancer samples. Measurements were repeated in MDA-MB-231 cells after MSI-1 and -2 siRNA-mediated double knockdown, with further experiments performed after MSI silencing. Flow cytometry helped quantify expression of CD44 and leukemia inhibitory factor receptor (LIFR), changes in apoptosis and cell cycle progression. Proliferation and irradiation-induced effects were assessed using colony formation assays. Radiation-related proteins were investigated via Western blots. Finally, cell invasion assays and digital holographic microscopy for cell migration were performed. MSI proteins showed strong correlations with Notch pathway elements. MSI knockdown resulted in reduction of stem cell marker expression, cell cycle progression and proliferation, while increasing apoptosis. Cells were radiosensitized as radioresistance-conferring proteins were downregulated. However, MSI -silencing-mediated LIFR downregulation resulted in enhanced cell invasion and migration. We conclude that, while MSI knockdown results in several therapeutically desirable consequences, enhanced invasion and migration need to be counteracted before knockdown advantages can be fully exploited.
Keywords	musashi rna-binding proteins ; breast cancer stem cells ; notch ; apoptosis ; proliferation ; radiotherapy ; egfr ; lifr ; migration ; invasiveness ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers.

Mathias Kaiser / Luisa Pohl / Steffi Ketelhut / Lena Kastl / Christian Gorzelanny / Martin Götte / Jürgen Schnekenburger / Francisco M Goycoolea / Björn Kemper

PLoS ONE, Vol 12, Iss 11, p e

2017 Volume 0187497

Abstract: We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem's influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) ... ...

Abstract	We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem's influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers.
Keywords	Medicine ; R ; Science ; Q
Subject code	500
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Quantitative stain-free and continuous multimodal monitoring of wound healing in vitro with digital holographic microscopy.

Dominik Bettenworth / Philipp Lenz / Philipp Krausewitz / Markus Brückner / Steffi Ketelhut / Dirk Domagk / Björn Kemper

PLoS ONE, Vol 9, Iss 9, p e

2014 Volume 107317

Abstract: Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure ... ...

Abstract	Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements.

Alexander Schwickert / Esther Weghake / Kathrin Brüggemann / Annika Engbers / Benjamin F Brinkmann / Björn Kemper / Jochen Seggewiß / Christian Stock / Klaus Ebnet / Ludwig Kiesel / Christoph Riethmüller / Martin Götte

PLoS ONE, Vol 10, Iss 12, p e

2015 Volume 0143993

Abstract: MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in ... ...

Abstract	MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3'-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer.
Keywords	Medicine ; R ; Science ; Q
Subject code	571
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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