Untersuchung der immunmodulatorischen Effekte von Pomalidomid auf retinale Zelllinien in vitro.

Die Retina mit ihrer physiologischen Abgrenzung durch die Blut-Retina-Schranke bildet ein sogenanntes immunprivilegiertes Organ, dessen einzige Immunzellen die Mikroglia darstellen. Diese Mikrogliazellen nehmen daher eine entscheidende Rolle bei der Schädigung und Degeneration der Netzhaut durch inflammatorische Prozesse sowohl bei hereditären als auch bei erworbenen Erkrankungen der Retina ein. Hierbei steht der Übergang der physiologisch ramifizierten Mikroglia in eine aktivierte, sogenannte reaktive Form und die dadurch folgende fehlregulierte Immunantwort mit Hyperinflammation und neuronaler Degeneration im Vordergrund. Da dies über einen gewissen Zeitraum hin zu einer Sehstörung im Rahmen der unterschiedlichen Erkrankungen führt, rückt die Mikroglia als möglicher Therapieansatz immer weiter in den Fokus der Betrachtung. Ziel der aktuellen Forschung ist es, eine möglichst potente Substanz für diese Zielsetzung zu finden. In dieser Arbeit wurde Pomalidomid, welches bereits in der Therapie des multiplen Myeloms eine immunmodulatorische Wirkung in der Tumorumgebung zeigt, hinsichtlich seiner immunmodulatorischen Effekte auf die retinale Mikroglia untersucht. Hierzu erfolgte die Betrachtung der Toxizität, der Genexpression und der Veränderungen der Morphologie in BV-2 Mikrogliazellen unter Einsatz von Pomalidomid sowie dessen Einfluss auf deren Effektorfunktionen wie Phagozytoseverhalten, Migrationsverhalten und Veränderungen der NO- Produktion. Zusätzlich wurde der Einfluss von Pomalidomid mittels konditionierten Mediums aus BV-2 Mikrogliazellen auf die Genexpression sowie die Morphologie von ARPE-19 Pigmentepithelzellen und dessen Einfluss auf das Apoptoseverhalten von ARPE-19 Pigmentepithelzellen sowie 661W Photorezeptorzellen untersucht. Es konnte gezeigt werden, dass der immunmodulatorische Effekt von Pomalidomid, der für Makrophagen des peripheren Bluts eindeutig beschrieben wurde, in der retinalen Mikroglia kaum vorhanden ist. Ein signifikanter Effekt durch Pomalidomid konnte ausschließlich in der Morphologie und dem Migrationsverhalten der BV-2 Mikrogliazellen sowie in der Genexpression durch ARPE-19 Zellen gezeigt werden. in der Untersuchung der Phagozytoserate zeigte sich eine Tendenz zur Reduktion dieser durch Pomalidomid. In allen weiteren Experimenten zeigte Pomalidomid keinen Einfluss. Aufgrund der unzureichenden antiinflammatorisch-immunmodulatorischen Wirkung von Pomalidomid auf die retinale Mikroglia sowie der verhältnismäßig hohen Toxizität der Substanz ist diese für einen Einsatz in der Therapie retinal- degenerativer Erkrankungen nicht vielversprechend, sodass der Fokus zukünftiger Forschung auf der Untersuchung alternativer Substanzen liegen sollte

Die sogenannte Rachentonsille, ihre Erkrankung und Behandlung

n/

Eduard Löri

n/

In Vitro Aging of Human Skin Fibroblasts: Age-Dependent Changes in 4-Hydroxynonenal Metabolism

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 \ub5M HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 \ub5M HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed

Frequency Distribution and Association of some Morpho- and Physiological Traits in Patients with Lung Diseases in Kosova

The aim of this study was to investigate the distribution of specific phenotypes in patients with lung diseases as well as their eventual association withthe risk of developing lung diseases. For this purpose 2777 patients with lung diseases and 2778 healthy individuals from all over Kosova were examined for the appearance of the following selected phenotypes: ear lobe free (ELF)/ ear lobe attached, normal chin (NC)/cleft chin, tongue roller (TR)/non roller, hand clasping right thumb over (HC)/ hand clasping left thumb over, righthanded (RH)/lefthanded. In addition, the blood group from ABO system and the presence or absence of the Rhesus factor as phenotypical markers were observed. The results obtained show significant differences between control and lung disease patients for NC (P≤0.05) and TR (P≤0.005) as well as for blood groups AB (P≤0.05) and O (P≤0.005). These results point to eventually increased levels of genetic load as a result of the increased homozygosity in some gene loci causing an increased frequency of some recessive phenotypes in patients with lung diseases. Together with the specific associations observed, these preliminary findings could serve as a basis for further in depth investigations with respect to the types of lung diseases, occupational exposure and dietary habits, and thus is expected to contribute to an understanding of predispositions and susceptibility to lung diseases

From Pinocytosis to Methuosis—Fluid Consumption as a Risk Factor for Cell Death

The volumes of a cell [cell volume (CV)] and its organelles are adjusted by osmoregulatory processes. During pinocytosis, extracellular fluid volume equivalent to its CV is incorporated within an hour and membrane area equivalent to the cell’s surface within 30 min. Since neither fluid uptake nor membrane consumption leads to swelling or shrinkage, cells must be equipped with potent volume regulatory mechanisms. Normally, cells respond to outwardly or inwardly directed osmotic gradients by a volume decrease and increase, respectively, i.e., they shrink or swell but then try to recover their CV. However, when a cell death (CD) pathway is triggered, CV persistently decreases in isotonic conditions in apoptosis and it increases in necrosis. One type of CD associated with cell swelling is due to a dysfunctional pinocytosis. Methuosis, a non-apoptotic CD phenotype, occurs when cells accumulate too much fluid by macropinocytosis. In contrast to functional pinocytosis, in methuosis, macropinosomes neither recycle nor fuse with lysosomes but with each other to form giant vacuoles, which finally cause rupture of the plasma membrane (PM). Understanding methuosis longs for the understanding of the ionic mechanisms of cell volume regulation (CVR) and vesicular volume regulation (VVR). In nascent macropinosomes, ion channels and transporters are derived from the PM. Along trafficking from the PM to the perinuclear area, the equipment of channels and transporters of the vesicle membrane changes by retrieval, addition, and recycling from and back to the PM, causing profound changes in vesicular ion concentrations, acidification, and—most importantly—shrinkage of the macropinosome, which is indispensable for its proper targeting and cargo processing. In this review, we discuss ion and water transport mechanisms with respect to CVR and VVR and with special emphasis on pinocytosis and methuosis. We describe various aspects of the complex mutual interplay between extracellular and intracellular ions and ion gradients, the PM and vesicular membrane, phosphoinositides, monomeric G proteins and their targets, as well as the submembranous cytoskeleton. Our aim is to highlight important cellular mechanisms, components, and processes that may lead to methuotic CD upon their derangement

Antioxidants / Effects of -carotene and its cleavage products in primary pneumocyte type II cells

-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8′carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of -carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of -carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 M. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations 1 M and 5 M, respectively, -carotene in the presence of DMNQ did not cause DNA damage. Instead, -carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of -carotene in the two cell types.(VLID)219514

NADPH oxidase mediates TNF-α-evoked in vitro brain barrier dysfunction: roles of apoptosis and time

The pro-inflammatory cytokine TNF-α severely perturbs the integrity of the blood–brain barrier (BBB). This study explored the specific roles of NADPH oxidase and associated downstream effectors by using human brain microvascular endothelial cells (HBMECs) and human astrocytes (HAs), the key components of BBB, alone or in co-cultures to mimic human BBB. Exposure to TNF-α (6 h) impaired BBB integrity as evidenced by marked decreases in transendothelial electrical resistance and concurrent increases in paracellular flux which appeared to subside with time (24 h). Increased barrier dysfunction concurred with increases in endothelial NADPH oxidase activity, O2radical dot− production, actin stress fibre formation, MMP-2/9 activities and concomitant decreases in antioxidant (CuZn-SOD and catalase) and tight junction (claudin-5 and occludin) protein expressions. Conversely, TNF-α did not affect astrocytic MMP activities and antioxidant enzyme expressions. Unlike BBB damage, rates of HBMEC and HA apoptosis increased by time. Suppression of NADPH oxidase by apocynin or diphenyleneiodonium prevented TNF-α-evoked morphological changes and apoptosis, attenuated endothelial MMP activity and helped retain usual tight junction protein expression and barrier function. In conclusion, HBMECs constitute the main source of oxidative stress and basement-membrane degrading endopeptidases in inflammatory conditions associated with excessive release of TNF-α where targeting NADPH oxidase may prove extremely beneficial in maintaining proper barrier activity through prevention of cytoskeletal and tight junction reorganisations

Hyperosmotic stress induces axl activation and cleavage in cerebral endothelial cells

Due to the relative impermeability of the blood-brain barrier many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the blood-brain barrier using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the blood-brain barrier. Previously we have shown that among targets of tyrosine phosphorylation are ?-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signaling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase- and proteasome-dependent and resulted in 50-55 kDa C-terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol-treated cells, therefore we assume that activation of Axl may be a protective mechanism against hypertonicity-induced apoptosis. Our results identify Axl as an important element of osmotic stress-induced signaling

Inhibition of TNF-α protects in vitro brain barrier from ischaemic damage

Cerebral ischaemia, associated with neuroinflammation and oxidative stress, is known to perturb blood–brain barrier (BBB) integrity and promote brain oedema formation. Using an in vitro model of human BBB composed of brain microvascular endothelial cells and astrocytes, this study examined whether suppression of TNF-α, a potent pro-inflammatory cytokine, might attenuate ischaemia-mediated cerebral barrier damage. Radical decreases in transendothelial electrical resistance and concomitant increases in paracellular flux across co-cultures exposed to increasing periods of oxygen-glucose deprivation alone (0.5–20 h) or followed by 20 h of reperfusion (OGD ± R) confirmed the deleterious effects of ischaemic injury on cerebral barrier integrity and function which concurred with reductions in tight junction protein (claudin-5 and occludin) expressions. OGD ± R elevated TNF-α secretion, NADPH oxidase activity, O2radical dot− production, actin stress fibre formation, MMP-2/9 activities and apoptosis in both endothelial cells and astrocytes. Increases in MMP-2 activity were confined to its extracellular isoform and treatments with OGD + R in astrocytes where MMP-9 could not be detected at all. Co-exposure of individual cell lines or co-cultures to an anti-TNF-α antibody dramatically diminished the extent of OGD ± R-evoked oxidative stress, morphological changes, apoptosis, MMP-2/9 activities while improving the barrier function through upregulation of tight junction protein expressions. In conclusion, vitiation of the exaggerated release of TNF-α may be an important therapeutic strategy in preserving cerebral integrity and function during and following a cerebral ischaemic attack