New advancements in highly sensitive time-resolved fluorescence two-photon microscopy: Theoretical approaches and bioscientific applications

A central methodological aim of the biosciences is to experimentally simulate, as good as possible, the real environmental conditions of biological systems in order to gain a true image of the effects and processes of interest. Thus, the bioscientific research is moving from test-tube observations towards in vivo investigations in intact cells and tissues. In this frame, the development of biophysical techniques, which allow an accurate and highly sensitive registration of changes in the studied system without disturbing the natural processes therein, is particularly significant. Especially the fluorescence laser microscopy provide a large diversity of versatile tools for bioscientific studies. In this work, two new theoretical approaches are developed, which allow an extended comprehension of the single-molecule fluorescence fluctuation microscopy based on two-photon excitation, one of the most sensitive techniques for biological investigations. Moreover, novel non-invasive fluorescence lifetime imaging experiments are performed, which enable us a detailed and accurate insight in the cellular NAD(P)H-metabolism and in the functionality of artificial skin as an organ.Eines der Hauptziele in den Biowissenschaften ist es, die tatsächliche Umgebung biologischer Systeme experimentell zu simulieren, um eine naturgetreue Abbildung der wichtigen Effekte und Prozesse in diesen Systemen zu erhalten. Aus diesem Grund entfernt sich die biowissenschaftliche Forschung von Untersuchungen im Reagenzglas und widmet zunehmend den Experimenten in intakten Zellen und Geweben Aufmerksamkeit. In diesem Zusammenhang ist die Entwicklung biophysikalischer Verfahren besonders wichtig, die eine sehr genaue und hoch-sensitive Aufnahme der Veränderungen in dem zu untersuchenden System erlauben, ohne dass die darin ablaufenden natürlichen Prozesse gestört werden. Besonders die Fluoreszenz-Laser-Mikroskopie stellt eine große Auswahl an Verfahren für biowissenschaftliche Untersuchungen zu Verfügung. In dieser Arbeit werden zwei neue theoretische Modelle vorgestellt, die ein detailliertes Verständnis der, auf Zwei-Photonen-Anregung basierenden, Einzelmolekül-Nachweis-Fluoreszenz-Fluktuations-Mikroskopie ermöglichen. Diese Methode stellt eine der empfindlichsten und genausten Methoden für biologische Anwendungen dar. Außerdem werden neuartige, gewebeschonende Fluoreszenz-Lebensdauer-Imaging Experimente vorgestellt, die eine umfangreiche und quantitative Einsicht in den NAD(P)H Zellmetabolismus und in die Funktionalität künstlicher Haut als Organ zulassen

In the Right Place, at the Right Time: Spatiotemporal Conditions Determining Plasma Cell Survival and Function

Plasma cells (PCs), the B lineage cells responsible for producing and secreting antibodies (Abs), are critical cellular components of the humoral immune system. While most of the antibody-secreting cells in the body have a rather short lifetime of a few days, some of them can become long-lived and persist in the body over the entire life span of an individual. The majority of these long-lived plasma cells secretes protective antibodies against pathogens, and are thereby crucial for the humoral component of immunological memory. The generation of these protective antibody-secreting cells can be triggered by an exposure to pathogens, and also by vaccination. Although the majority of plasma cells are protective, sometimes long-lived plasma cells produce autoreactive antibodies, which contribute to the pathogenesis and perpetuation of chronic autoimmune diseases, including lupus erythematosus, rheumatoid arthritis, or multiple sclerosis. In order to promote the formation of protective antibody-secreting cells and to target pathogenic plasma cells, it is crucial to understand the signals which promote their longevity and allow them to exert their function. In recent years, it has become clear that plasma cells depend on extrinsic factors for their survival, leading to the concept that certain tissue microenvironments promote plasma cell retention and longevity. However, these niches are not static structures, but also have dynamic features with respect to their cellular composition. Here, we review what is known about the molecular and cellular composition of the niches, and discuss the impact of dynamic changes within these microenvironments on plasma cell function. As plasma cell metabolism is tightly linked to their function, we present new tools, which will allow us to analyze metabolic parameters in the plasma cell niches in vivo over time

In vitro vascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge of in vitro vascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineered in vitro tissue constructs

Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet’s intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring

Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE

Most multiple sclerosis (MS) patients develop over time a secondary progressive disease course, characterized histologically by axonal loss and atrophy. In early phases of the disease, focal inflammatory demyelination leads to functional impairment, but the mechanism of chronic progression in MS is still under debate. Reactive oxygen species generated by invading and resident central nervous system (CNS) macrophages have been implicated in mediating demyelination and axonal damage, but demyelination and neurodegeneration proceed even in the absence of obvious immune cell infiltration, during clinical recovery in chronic MS. Here, we employ intravital NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX1–4, DUOX1, 2) and, thus, to identify the cellular source of oxidative stress in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) in the remission phase of the disease. This directly affects neuronal function in vivo, as monitored by cellular calcium levels using intravital FRET–FLIM, providing a possible mechanism of disease progression in MS

Teriflunomide preserves peripheral nerve mitochondria from oxidative stress-mediated alterations

Mitochondrial dysfunction is a common pathological hallmark in various inflammatory and degenerative diseases of the central nervous system, including multiple sclerosis (MS). We previously showed that oxidative stress alters axonal mitochondria, limiting their transport and inducing conformational changes that lead to axonal damage. Teriflunomide (TFN), an oral immunomodulatory drug approved for the treatment of relapsing forms of MS, reversibly inhibits dihydroorotate dehydrogenase (DHODH). DHODH is crucial for de novo pyrimidine biosynthesis and is the only mitochondrial enzyme in this pathway, thus conferring a link between inflammation, mitochondrial activity and axonal integrity. Here, we investigated how DHODH inhibition may affect mitochondrial behavior in the context of oxidative stress. We employed a model of transected murine spinal roots, previously developed in our laboratory. Using confocal live imaging of axonal mitochondria, we showed that in unmanipulated axons, TFN increased significantly the mitochondria length without altering their transport features. In mitochondria challenged with 50 µM hydrogen peroxide (H2O2) to induce oxidative stress, the presence of TFN at 1 µM concentration was able to restore mitochondrial shape, motility, as well as mitochondrial oxidation potential to control levels. No effects were observed at 5 µM TFN, while some shape and motility parameters were restored to control levels at 50 µM TFN. Thus, our data demonstrate an undescribed link between DHODH and mitochondrial dynamics and point to a potential neuroprotective effect of DHODH inhibition in the context of oxidative stress-induced damage of axonal mitochondria