Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degreesC to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P<0.01) in early lactating cows (15.1%, n=9) compared with midlactating cows (5.3%, n=10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n=7) and midlactating cows (34.0%, n=8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n=7) was significantly higher than that in midlactating cows (14.2%, n=8) (P<0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis

High content image-based cytometry as a tool for nuclear fingerprinting

Cytomics aims at understanding the functional relationships between cellular phenotypes (cytome) and metabolic pathways (proteome) that result from a combination of genetically defined mechanisms (genome) and environmental conditions [1,2]. Although flow-cytometry is able to measure the optical properties of single cells at a rate of >1000 cells per minute it has a limited capability of mapping individual events. To accurately quantify (sub-) cellular characteristics within a natural context there is a fast-growing need for image-based cytometry. Images, obtained with fluorescence microscopy, provide the exact information on signal intensity, location and distribution of specific molecules within intact cell systems (tissue or monolayers) and allow for investigating cellular properties in relation to the cell-ecological context [3]. Previously, we have developed a cytometric approach for scoring DNA lesion endpoints in confocal images of murine fibroblasts [3]. We now present a generalized approach for multivariate phenotypic profiling of individual nuclei using automated fluorescence mosaic microscopy and optimized digital image processing tools. An indefinite number of fields, z-slices and channels can be analyzed; the only prerequisite is the presence of a nuclear counterstain, which is used for the generation of masks. To anticipate for erroneous segmentation of clustered nuclei in dense cell cultures we implemented an iterative conditional segmentation (ics) algorithm that uses both morphological and intensity information from the image (Figure 1). The method makes use of a priori knowledge about the size and shape of nuclei in stringent feedback selection of correctly segmented nuclei. Depending on the degree of clustering, segmentation performance varies between 95% and 100%. Complete analysis of nuclei and subnuclear features for a region of 25 images of 1000x1000 pixels, 3 z-slices and 3 channels only takes ~ 3 minutes or ~ 0.7sec/nucleus. Our method is insensitive to scaling, illumination heterogeneity and variability or non-uniformity of staining. We have successfully applied our system in cell cycle analysis, scoring of transfection efficiency and assessment of (localized) DNA damage in response to genotoxic stress and ionizing radiation

Apoptosis of bovine neutrophils following diapedesis through a monolayer of endothelial and mammary epithelial cells.

In a two-chamber system, isolated blood polymorphonuclear neutrophil leukocytes (PMN) were allowed to migrate (5 h, 37 C) in response to bovine complement component C5a across calfskin and rat-tail type I collagen-coated micropore membranes, arterial endothelial, or mammary epithelial cell monolayer on calfskin and rat-tail collagen-coated membranes, respectively. Migration through calfskin collagen-coated membranes resulted in 14.5% +/- 3.4% apoptotic PMN, which was significantly higher than 6.6% +/- 1.2% apoptotic nonmigrated C5a-treated PMN. The addition of an endothelial or epithelial cell monolayer to collagen-coated membranes prevented apoptosis of migrated PMN. After removing the membranes, nonmigrated (untreated and C5a treated) and migrated PMN were incubated for an additional 20 h. At this time point, 69.1% +/- 4.5% and 47% +/- 4.5% of PMN that have migrated through a calfskin-coated membrane and an endothelial monolayer, respectively, were apoptotic, compared with 28.2% +/- 3.0% and 21.1% +/- 4.5% apoptotic untreated and C5a-treated PMN, respectively; 46.9% +/- 4.8% of PMN that have migrated through rat-tail-coated membranes were apoptotic compared with 14.7% +/- 2.3% and 9.3% +/- 1.2% apoptotic untreated and C5a-treated PMN, respectively. Migration across rat-tail collagen-coated membranes with a monolayer of epithelial cells did not affect apoptosis of migrated PMN, even after 20 h of incubation. In conclusion, migration of PMN across collagen-coated membranes (either calfskin or rat-tail collagen) induced an apoptotic response, which was downregulated by a monolayer of endothelial cells and was negated by an epithelial cell monolayer

Multilevel analysis of nuclear dynamics in lamin perturbed fibroblasts

The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression [1]. Most studies regarding the mechanical properties of the nucleus in laminopathic conditions are based on the induction of extracellular stress, such as strain or compression, and focus on nuclear integrity and/or nucleo-cytoskeletal interaction [2]. Far less is known about the role of nuclear organization and mobility under basal steady-state conditions. In this study, we quantitatively compared nuclear organization, nuclear deformation and chromatin mobility of fibroblasts from a Hutchinson-Gilford progeria patient with cells from a lamin A/C-deficient patient and wild-type dermal fibroblasts. To this end, we created a toolbox in imageJ for automatically analyzing both nuclear as well as subnuclear dynamics in living cells. Simultaneously, we developed a workflow for comparing cellular morphology and subcellular protein distribution in a high content fashion. We found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the seconds time scale. In contrast, progeria cells showed overall reduced nuclear dynamics. In addition, high content analysis revealed marked morphological and topological differences between different culture passages within a cell type and between different pathological variants of culture-age matched laminopathic cell types

The sublime in the visual culture of the Seventeenth-Century Dutch Republic

Contrary to what Kant believed about the Dutch (and their visual culture) as “being of an orderly and diligent position” and thus having no feeling for the sublime, this book argues that the sublime played an important role in seventeenth-century Dutch visual and decorative art, architecture, and theater.By looking at different visualizations of exceptional heights, divine presence, overwhelming natural phenomena, political grandeur, extreme violence, and extraordinary artifacts, the authors demonstrate how viewers were confronted with the sublime, which evoked in them a combination of contrasting feelings of awe and fear, attraction and repulsion. In studying seventeenth-century Dutch visual culture through the lens of notions of the sublime, we can move beyond the traditional and still widespread views on Dutch art as the ultimate representation of everyday life and the expression of a prosperous society in terms of calmness, neatness, and order.The book will be of interest to scholars working in art history, visual culture, architectural history, and cultural history.Horizon 2020(H2020)Medieval and Early Modern Studie