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- Department of Biomedical Imaging, National Cerebral and Cardiovascular Research Center, Suita, Japan (2)
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- Institut for Molecular Biology and CMBI, Department of Genomics, Stem Cell Biology and Regenerative Medicine, Leopold-Franzens-University Innsbruck, Innsbruck, Austria (2)
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- Johns Hopkins School of Medicine, Baltimore, MD, U.S. (1)
Rat organic cation transporter 1 (rOCT1): investigation of conformational changes and ligand binding
(2008)
Polyspecific organic cation transporters (OCTs) of the SLC22 family mediate downhill transport of organic cations and play an essential role in excretion and distribution of endogenous organic cations and for the uptake, elimination and distribution of cationic drugs and toxins. Although physiological and pharmacological significance of OCTs is widely accepted, many questions concerning structure and transport mechanism still remain open. To investigate conformational changes of the rat OCT1 during transport cycle, voltage-clamp fluorometry was performed with a cysteine-deprived mutant in which phenylalanine 483 in transmembrane helix (TMH) 11 close to the extracellular surface was replaced by cysteine and covalently labeled with tetramethylrhodamine-6-maleimide. Potential-dependent fluorescence changes were observed that were sensitive to the presence of substrates choline, tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPP), and of the contransported inhibitor tetrabutylammonium (TBuA). The data suggest that the transporter undergoes conformational changes in voltage- and substrate-dependent manner which are compatible with alternating access mechanism. Using potential-dependent fluorescence changes as readout, one high-affinity binding site per substrate and two highaffinity binding sites for TBuA were identified in addition to the previously described single interaction sites. Coexisting high-affinity cation binding sites in organic cation transporters may collect xenobiotics and drugs; however, translocation of organic cations across the membrane may only be induced when a low-affinity cation binding site is loaded. Whereas high-affinity binding of TBuA has no effect on cation uptake by wildtype rat OCT1, replacement by cysteine or serine of amino acids W147, F483, and F486 located in a modeled contact region between TMH2 and TMH11 outside the binding pocket leads to inhibition of MPP or TEA uptake. Thus, mutations of amino acids in transport relevant key positions, which can be distinct from the cation binding region, may transform noninhibitory highaffinity binding sites of high-affinity inhibition sites and thereby cause adverse drug reactions in patients.
Als Therapieversuch bei Plexusläsionen wird die Replantation ausgerissener Vorderwurzelfasern durchgeführt. Voraussetzung für die erfolgreiche Regeneration von Motoneuronaxonen sind 1. Überleben einer ausreichenden Anzahl von Motoneuronen 2. erfolgreiche Wiederherstellung der Kontuität ausgerissener Axone mit dem Rückenmark und 3. funktionelle Hochwertigkeit regenerierter Axone. Neurotrophe Faktoren können Überleben und Regenerationsfähigkeit von Motoneuronen fördern. Gegenstand der vorliegenden Arbeit war die Analyse des Einflusses von CNTF und BDNF auf die Regeneration von Motoneuronaxonen nach Ausriss und Replantation im Segment C7 nach einer Überlebenszeit von 3 Wochen bzw. 6 Monaten. Vervollständigt wurden diese Untersuchungen durch detaillierte morphologische Analysen von Spinalganglien, durchtrennter Hinterwurzel und verletztem Hinterhorn. In verschiedenen Gruppen von adulten Kaninchen wurden CNTF, BDNF, oder beide Faktoren auf die ventrolaterale Replantationsstelle appliziert, Kontrollen wurden ohne Faktor belassen (n>5). Die Überlebenszeit der Versuchstiere lag bei 3 Wochen (n=3 Kontrollen) und 6 Monaten (n=27). Aus dem perfundiertem Gewebe wurden Semidünnschnitte durch Vorderwurzel/Spinalganglien und Kryostatserienschnitte durch das Segment C7 angefertigt. DiI-Fluoreszenztracing, Markscheidenfärbung, eine modifizierte Klüver-Barrera-Färbung der Kryostatschnitte sowie eine Touloidinblaufärbung der Semidünnschnitte ermöglichte die morphologische und morphometrische Analyse des Gewebes. Die Anzahl der überlebenden Motoneurone lag nach sechs Monaten bei allen Versuchsgruppen bei etwa 30%. Fluoreszenz-Tracing und Markscheidenfärbungen von Serienschnitten zeigten, dass Axone sowohl über die ursprünglichen ventralen Austrittstellen als auch über die ventrolaterale Replantationsstelle das Rückenmark verließen und im Bereich des Spinalganglions eine kompakte Vorderwurzel bildeten. Ventral austretende Axone zeigten signifikant größere Durchmesser als lateral austretende. Ausmaß und Art der Regeneration waren interindividuell unterschiedlich, die besten Ergebnisse zeigte die Replantation nah am ursprünglichen Austrittsort der Vorderwurzel. Unterschiede zwischen den Gruppen waren nicht deutlich. In Semidünnschnitten durch die regenerierte Vorderwurzel fanden sich nach drei Wochen kaum intakte, myelinisierte Axone, nach sechs Monaten war die Zahl der Axone auf etwa 45% der Zahl der gesunden Seite angestiegen. Regenerierte Axone waren dünn, typische Motoneuronaxone stellten nur einen kleinen Teil der regenerierten Axone. Gruppenunterschiede fanden sich im Axon-Myelinverhältnis, das bei Kontrollen der replantierten Seiten signifikant erniedrigt war. Diese Erniedrigung war noch vorhanden, jedoch nicht mehr signifikant bei Tieren, die mit CNTF- und BDNF-behandelt wurden. Die replantierten Vorderwurzeln der CNTF+BDNF-Gruppe zeigte überwiegend eine signifikant bessere Myelinisierung als die replantierten Kontrollen. An der früheren Hinterwurzeleintrittszone am Rückenmark wurden in Tieren mit geringem Verletzungsausmaß kleine ZNS-Gewebsprotrusionen beobachtet, in denen sich myelinisierte Axone befanden. Diese Axone zeigten eine Wachstumsrichtung in die Peripherie, was auf eine Sprossung der sensorischen Rückenmarksneurone schließen lässt. Innerhalb des Spinalganglions waren Neuron- und Axondichte auf den verletzten Seiten nicht wesentlich verändert. Eine leichte Abnahme des relativen Anteils großer Neurone und Axone wurde in den verletzten Seiten der Kontrollgruppe beobachtet. Für Axone war diese Abnahme statistisch signifikant. Im Gegensatz dazu war dies in Tieren, die mit neurotrophen Faktoren behandelt wurden, nicht zu beobachten. Bei allen Tieren zeigte sich ein beträchtliches Auswachsen von Hinterwurzelaxonen aus dem Spinalganglion. Diese Axone fanden keine spontane Verbindung mit dem proximalen Rest der Wurzel, sondern waren durch Bindegewebe eingehüllt. Bei etwa der Hälfte der Tiere zeigte sich, dass einer Untergruppe dieser Axone in Richtung des Narbengewebes der replantierten Vorderwurzel gewachsen war und über Defekte in der Bindegewebshülle teilweise sogar in die Vorderwurzel einwuchsen. Ein möglicher Einfluss der applizierten neurotrophen Faktoren auf das quantitative Regenerationsergebnis scheint also in diesem Modell gering zu sein. Auf eine qualitative Verbesserung deutet die Normalisierung des Axon-Myelinverhältnisses großer regenerierter Axone bei Kombinationsbehandlung hin. Die im vorliegenden Modell beträchtliche Regenerationskapazität der Hinterwurzel scheint bisher unterschätzt worden zu sein. Das unerwartete Einwachsen von Hinterwurzelaxonen in die Vorderwurzel könnte mit einer funktionellen Beeinträchtigung der regenerierten Vorderwurzel verbunden sein.
A growing body of scientific evidence indicates that protein homeostasis, also designated as proteostasis, is causatively linked to chronic diabetic nephropathy (DN). Experimental studies have demonstrated that the insulin signaling in podocytes maintain the homeostatic unfolded protein response (UPR). Insulin signaling via the insulin receptor non-canonically activates the spliced X-box binding protein-1 (sXBP1), a highly conserved endoplasmic reticulum (ER) transcription factor, which regulates the expression of genes that control proteostasis. Defective insulin signaling in mouse models of diabetes or the genetic disruption of the insulin signaling pathway in podocytes propagates hyperglycemia induced maladaptive UPR and DN. Insulin resistance in podocytes specifically promotes activating transcription factor 6 (ATF6) dependent pathogenic UPR. Akin to insulin, recent studies have identified that the cytoprotective effect of anticoagulant serine protease-activated protein C (aPC) in DN is mediated by sXBP1. In mouse models of DN, treatment with chemical chaperones that improve protein folding provides an additional benefit on top of currently used ACE inhibitors. Understanding the molecular mechanisms that transmute renal cell specific adaptive responses and that deteriorate renal function in diabetes will enable researchers to develop new therapeutic regimens for DN. Within this review, we focus on the current understanding of homeostatic mechanisms by which UPR is regulated in DN.
Since exposure therapy for anxiety disorders incorporates extinction of contextual anxiety, relapses may be due to reinstatement processes. Animal research demonstrated more stable extinction memory and less anxiety relapse due to vagus nerve stimulation (VNS). We report a valid human three-day context conditioning, extinction and return of anxiety protocol, which we used to examine effects of transcutaneous VNS (tVNS). Seventy-five healthy participants received electric stimuli (unconditioned stimuli, US) during acquisition (Day1) when guided through one virtual office (anxiety context, CTX+) but never in another (safety context, CTX−). During extinction (Day2), participants received tVNS, sham, or no stimulation and revisited both contexts without US delivery. On Day3, participants received three USs for reinstatement followed by a test phase. Successful acquisition, i.e. startle potentiation, lower valence, higher arousal, anxiety and contingency ratings in CTX+ versus CTX−, the disappearance of these effects during extinction, and successful reinstatement indicate validity of this paradigm. Interestingly, we found generalized reinstatement in startle responses and differential reinstatement in valence ratings. Altogether, our protocol serves as valid conditioning paradigm. Reinstatement effects indicate different anxiety networks underlying physiological versus verbal responses. However, tVNS did neither affect extinction nor reinstatement, which asks for validation and improvement of the stimulation protocol.
Rekrutierung von Stromazellen aus gefäßwandresidenten Vorläuferzellen während der Tumorgenese
(2021)
Tumore bestehen nicht nur aus malignen Zellen, sondern ebenfalls aus einer Vielzahl an nicht tumorigenen Zellen, die den Tumor auf vielfältige Weise unterstützen und den Tumor vor therapeutischen Maßnahmen schützen. Die Frage der Herkunft dieser Zellen insbesondere in einem nicht vaskularisierten Tumor ist daher auch für die Entwicklung zukünftiger Therapeutika relevant. In dieser Arbeit wurde eine Methode etabliert, die im dreidimensionalen Raum die Untersuchung des Einflusses von Tumorzellen auf die vaskuläre Adventitia am Model der Mausaorta ermöglicht. Dazu erfolgte die Einbettung von Alginatbeads aus verschiedenen Tumorzelllinien in eine gemeinsame Kollagenmatrix mit murinen Aortenringen. Während des zehntägigem Versuchszeitraums wurde die Aussprossung von Zellen aus den Aortenringen beobachtet und quantifiziert. Es wurde festgestellt, dass die Auswanderung während des Versuchszeitraums zunimmt und dass die Konfrontation mit der Zytokinmischung der Tumorzellen zu einer stärkeren Aussprossung führt, als die Stimulation mit VEGF oder keine Stimulation. Eine gerichtete Auswanderung der Zellen in Richtung der Tumorbeads konnte nicht nachgewiesen bzw. bestätigt werden. Kapilläre Aussprossungen waren nur in geringem Ausmaß zu beobachten. Bei Charakterisierung der ausgewanderten Zellen mittels immunhistochemischer Färbungen waren keine F4/80-positiven und nur einzelne CD34-positive Zellen zu finden. CD31-positive Endothelzellen stellten die Mehrheit der ausgewanderten Zellen bei Tumorzellkonfrontation. Perizyten, die mit dem Marker NG2 gefärbt wurden, stellten eine Mehrheit der migrierten Zellen bei allen Bedingungen.
Die in dieser Arbeit etablierte Methode des Aortenring-Bead-Konfrontationsassays ermöglicht es, in Echtzeit den Einfluss von Tumorzellen auf die Gefäßwand im dreidimensionalen Raum zu beobachten. Der Aortenring-Bead-Konfrontationsassay bietet eine Vielzahl an Variationsmöglichkeiten und stellt daher eine vielversprechende Möglichkeit dar, die Lücke zwischen zweidimensionalen in vitro-Experimenten und kostenintensiven in vivo-Versuchen zu schließen.
Reorganisation der Zellkontakte der Endothelbarriere bei der Stabilisierung durch cAMP und Rac1
(2012)
Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen.
Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt.
In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet.
In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine.
Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden.
Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam.
Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen.
Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44—a marker of acinar cells—diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.
Ca2+ dependent cell adhesion molecules (cadherins) are central for a variety of cell and tissue functions such as morphogenesis, epithelial and endothelial barrier formation, synaptic function and cellular signaling. Of paramount importance for cadherin function is their specific extracellular adhesive trans-interaction. Cadherins are embedded in a cellular environment of intracellular and extracellular regulators that modify cadherin binding in response to various physiological and pathological stimuli. Most experimental approaches used for studying cadherin interaction however lack a physiological proof of principle mostly by not investigating cadherins in their physiological environment. In the present cumulative dissertation, experimental approaches were applied to characterize and modulate vascular endothelial (VE)-cadherin and desmocadherin functions in the (patho-)physiological contexts of endothelial permeability regulation and disturbance of epidermal barrier function, which is typical to the blistering skin disease pemphigus, respectively. Whereas VE-cadherin is a key regulator of the endothelial barrier that separates the blood compartment from the interstitial space of tissues, desmosomal cadherins are crucial for maintenance of epidermal integrity and separation of the external environment from the body’s internal milieu. Cadherin functions were both investigated in cell-free and cell-based conditions: by using biophysical single molecule techniques like atomic force microscopy (AFM), cadherin function could be investigated in conditions, where contributions of intracellular signaling were excluded. These experiments were, however, compared and combined with cell-based experiments in which cadherins of epidermal or endothelial cell cultures were probed by laser force microscopy (laser tweezers), fluorescence recovery after photobleaching (FRAP) and other techniques. The autoimmune blistering skin diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are caused by autoantibodies directed against the extracellular domains of the desmosomal cadherins desmoglein (Dsg) 1 and 3, which are important for epidermal adhesion. The mechanism of autoantibody-induced cell dissociation (acantholysis) in pemphigus, however, is still not fully understood. For the first time, it is shown by AFM force spectroscopy that pemphigus autoantibodies directly inhibit Dsg3 adhesion by steric hindrance but do not inhibit adhesion of Dsg1. However, the full pathogenicity of the autoantibodies depended on cellular signaling processes, since autoantibodies targeting Dsg1 also resulted in loss of cadherin-mediated adhesion in cell-based experiments. However, two other signaling pathways that have been reported to be involved in pemphigus pathogenesis, i.e. epidermal growth factor receptor (EGFR) and c-Src activation, were not found to be important in this context. Furthermore, peptide-based modulators of cadherin functions were generated for Dsg1/3 and VE-cadherin. By comparing Dsg1, Dsg3 and VE-cadherin sequences to published X-ray structures of cadherin trans-interactions, specific amino acid sequences of the binding pockets of these cadherins were identified. Peptide versions of these motifs were synthesized and the antagonistic functions of these “single peptides” were validated by AFM force spectroscopy as well as by cell-based assays. By linking two single peptides in tandem, stabilization of cadherin bonds because of by cross-bridge formation between trans-interacting cadherins was demonstrated. Protective effects of tandem peptides were shown by partly preventing pemphigus autoantibody-induced acantholysis, or in the case of VE-cadherin, by stabilizing endothelial barrier properties against barrier disrupting agents like the Ca2+ ionophore A23187 and an inhibitory VE-cadherin antibody. Most importantly, VE-cadherin tandem peptides abolished microvascular hyperpermeability induced by the physiologic inflammatory agent tumor necrosis factor-α in the rat mesentery in vivo. Both classes of tandem peptides therefore can be considered as a starting point for the generation of potential therapeutic agents that might prevent cell dissociation in pemphigus and breakdown of the endothelial barrier under inflammatory conditions.
The Na+-D-glucose cotransporter in small intestine is regulated in response to food composition. Short term regulation of SGLT1 occurs post-transcriptionally in response to changes in luminal glucose. Adaptation to dietary carbohydrate involves long term regulation at the transcriptional level. The intracellular protein RS1 (gene RSC1A1) is involved in transcriptional and post-transcriptional regulation of SGLT1. RS1 contains an N-terminal domain with many putative phosphorylation sites. By Expressing SGLT1 in oocytes of Xenopus laevis it was previously demonstrated that the post-transcriptional down-regulation of SGLT1 by RS1 was dependent on the intracellular glucose concentration and activated by protein kinase C (PKC). The role of RS1 for short term regulation of SGLT1 in mouse small intestine in response to glucose and PKC was investigated comparing effects in RS1-/- mice and wildtype mice. Effects on SGLT1 activity were determined by measuring phlorizin inhibited uptake of α-methylglucoside (AMG). The involvement of RS1 in glucose dependent short term regulation could not be elucidated for technical reasons. However, evidence for RS1 independent short-term downregulation of SGLT1 after stimulation of PKC could be provided. It was shown that this downregulation includes decrease in the amount and/or in turnover of SGLT1 in the brush-border membrane as well as an increase of substrate affinity for AMG transport. Trying to elucidate the role of RS1 in long term regulation of SGLT1 in small intestine in response to glucose and fat content of the diet, wildtype and RS1-/- mice were kept for 2 months on a normo-caloric standard diet with high glucose and low fat content (ND), on a hyper-caloric glucose-galactose reduced diet with high fat content (GGRD) or on a hyper-caloric diet with a high fat and high glucose content (HFHGD). Thereafter the animals were starved overnight and SGLT1 mediated AMG uptake was measured. Independent of diet AMG uptake in ileum was smaller compared to duodenum and jejunum. In jejunum of wildtype and RS1-/- mice kept on the fat rich diets (GGRD and HFHGH) transport activity of SGLT1 was lower compared to mice kept on ND with low fat content. This result suggests an RS1 independent downregulation due to fat content of diet. Different to RS1-/- mice, the duodenum of wildtype mice showed transport activity of SGLT1 smaller in mice kept on glucose galactose reduced diet (GGRD) compared to the glucose galactose rich diets (ND and HFHGG). These data indicate that RS1 is involved in glucose dependent long term regulation in duodenum.
Rubus admirabilis Drenckhahn ist eine tetraploide neue Brombeerart der Rubus-Sektion Rubus, Serie Vestiti. Die Stängelblätter sind 5-zählig, hand- bis schwach fußförmig geteilt mit länglich obovaten, zugespitzen Endblättchen und anliegend behaarter Oberseite und hellgrün schimmernder, fühlbar weich behaarter Unterseite. Die Schösslinge sind mäßig bogig (bis zu 2m), teilweise kletternd, stumpfwinklig, graugrün bis stumpfbraun, dicht behaart mit zahlreichen gestielten Drüsen und Borsten. Stachel: 7−15/5 cm, schlank, 4−6mm lang, gerade, überwiegend 30-45º geneigt. Die Blütenstiele sind dicht behaart (abstehend und teilweise anliegend), mit 2−4/cm schlanken, geraden bis leicht gekrümmten Stachelchen (1−2 mm lang) und zahlreichen gestielten Drüsen (0,3−0,5 mm lang) sowie einigen Borsten. Die Art ist schattentolerant und bevorzugt feuchte Böden. Der Typusfundort ist wahrscheinlich der Ansiedlungs- oder Ursprungsort der Art. Er liegt westlich der Stadt Garding auf der nordfriesischen Halbinsel Eiderstedt (mehrere hundert Sträucher und Gebüsche). Dort wurden im Zuge einer Aufforstung 1970 mehrere nichtheimische Brombeerarten eingeschleppt. Rubus admirabilis hat sich südlich bis Heide in Dithmarschen und nördlich bis Amrum ausgebreitet (maximaler Arealdurchmesser von 70 km) und befindet sich in einer expansiven Phase.
Rubus pseudoglotta Drenckhahn & W. Jansen ist eine tetraploide Brombeerart aus der Sektion Corylifolii (Serie Subradula), die bisher zum Variabilitäts-Spektrum von R. phylloglotta (Frid.) Å. Gust. gezählt wurde. Charakteristische Merkmale sind die 4 (3–5)-zähligen Blätter mit obovaten Endblättchen mit kurzer (ca. 1 cm) abgesetzter Spitze, kurzhaariger Blattoberseite und fühlbar behaarter grüner Blattunterseite. Die flach bogigen, teils klimmenden Schösslinge sind überwiegend stumpfkantig, grün bis rötlichbraun, schwach behaart und reichlich mit 2–4 (5) mm langen, geraden bis schwach gekrümmten Stacheln und kleineren Stacheln, Stachelhöckern, Stieldrüsen und Borsten besetzt. Die Blütenstiele sind mit 2–8 (pro cm) schlanken, geraden bis leicht gekrümmten Stacheln (1–2 mm lang) und zahlreichen Stieldrüsen (teils bis 0,6 mm lang) besetzt. Die Sippe wächst bevorzugt an Straßen- und Wegrändern und in Hecken. Die bekannt gewordenen Fundstellen erstrecken sich von Rendsburg bis in das Umfeld von Kiel, nordwärts bis zu den dänischen Inseln Alsen und Fünen. Unsere Untersuchungen zeigen weiterhin, dass R. phylloglotta bisher nicht in Schleswig-Holstein/Deutschland nachgewiesen wurde. Ob R. phylloglotta überhaupt außerhalb der Insel Tåsinge in Dänemark vorkommt, bedarf weiterer Nachforschungen.
Rubus viridilucidus Drenckhahn ist eine tetraploide Brombeerart (2n=28) aus der Sektion Corylifolii, Serie Subcanescentes mit einem Genomgewicht (2C-Wert) von 1,49 pg, das dem Genomgewicht verwandter Sippen der Serie Subcanescentes wie R. scabrosus, R. fasciculatiformis und R. fasciculatus (1,52–1,54 pg) aus Unterfranken entspricht. Charakteristische Merkmale sind 3–4(5)-zählige Blätter mit herab gekrümmten rundlichen bis breit obovaten Endblättchen und breitovalen Seitenblättchen, die eine völlig unbehaarte, lichtgrüne, mattglänzende Blattoberfläche besitzen mit kontrastierender hell grünlich-grauer, samtig behaarter Blattunterseite. Die überwiegend rundlichen bis stumpf kantigen, lichtgrünen bis rötlich überlaufenen Schösslinge sind unbehaart und spärlich mit kurzen (<4mm) nadelförmigen Stacheln und wenigen Stieldrüsen besetzt. R. viridilucidus entwickelt zusätzlich zu den Blütenzweigen der zweijährigen Schösslinge (Ausbreitungsschösslinge) einen besonderen blühenden 0,8 bis 1,6 m langen Schösslingstyp aus, den Rispenschössling, der direkt aus dem Wurzelstock entspringt und terminal in eine Blütenrispe ausläuft. Bei R. viridilucidus sind zwei verschiedene Typen von Rispenschösslingen ausgebildet. Die Sippe wächst bevorzugt auf gestörten Flächen wie Brachen, Straßenrändern, Lagerplätzen, Weinbergrändern und kann sich mit 1–2 m jährlichem Zuwachs (Satellitenbildauswertung, Vermessungen vor Ort) schnell ausbreiten. Die bekannt gewordenen Fundstellen erstrecken sich vom nördlichen Baden-Württemberg bis in den nördlichsten Teil von Bayern (Rhön).
SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.
The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.
Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication.
Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform.
Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis.
Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks.
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30–40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.
Die Arten des Schweizer Ranunculus-auricomus-Komplexes sind nur zu einem Teil bekannt. Zur vollständigeren Erfassung des Komplexes wurden Exkursionen in die südwestliche und östliche Schweiz unternommen. Es wurden sieben neue Arten entdeckt, die hier beschrieben und abgebildet sind. Ihre Taxonomie und Gefährdung wird diskutiert. R. chalarocarpus W. Koch ex Dunkel ist bereits bei Koch provisorisch erwähnt, R. clavicornis Dunkel wird nun gültig beschrieben. Beide Arten sind aufgrund ihres Vorkommens in Auwäldern und feuchten Laubwäldern stark gefährdet, R. clavicornis sogar fast ausgestorben. Der neu beschriebene R. thurgoviae kommt im Osten der Schweiz vor (Kanton Thurgau). Die bislang bekannte Verbreitung von R. allobrogorum Dunkel, R. crenulatus Dunkel, R. genevensis Dunkel und R. lineatus ist fast vollständig auf den Kanton Genf beschränkt. Die Arten des Ranunculus auricomus-Komplexes sind ein sensibler Indikator für Veränderungen der Vegetation und Umwelt und sollten diesbezüglich deutlich mehr Gewicht bekommen.
Macrophages predominate the inflammatory landscape within multiple sclerosis (MS) lesions, not only regarding cellularity but also with respect to the diverse functions this cell fraction provides during disease progression and remission. Researchers have been well aware of the fact that the macrophage pool during central nervous system (CNS) autoimmunity consists of a mixture of myeloid cells. Yet, separating these populations to define their unique contribution to disease pathology has long been challenging due to their similar marker expression. Sophisticated lineage tracing approaches as well as comprehensive transcriptome analysis have elevated our insight into macrophage biology to a new level enabling scientists to dissect the roles of resident (microglia and non-parenchymal macrophages) and infiltrating macrophages with unprecedented precision. To do so in an accurate way, researchers have to know their toolbox, which has been filled with diverse, discriminating approaches from decades of studying neuroinflammation in animal models. Every method has its own strengths and weaknesses, which will be addressed in this review. The focus will be on tools to manipulate and/or identify different macrophage subgroups within the injured murine CNS.
Primary infection of HEp-2 cells with rubella virus resulted in non-cytophatic longterm persistent infection. During four years of persistence the virus was produced in sufficient quantities (up to 6 logs PFU/ml) and did not differ from the parental variant in its pathogenicity for BHK-21 or RK-13 cells, or hemagglutinating activity, but formed smaller plaques. Persistent virus preserved the original antigenicity as judged from reciprocal hemagglutination-inhibition or plaque reduction-neutralization tests with polyclonal antisera. Both original and persistent rubella viruses were thermoresistant (T 56° C) and sligthly temperature-sensitive. Clonal analysis revealed presence of ts-mutants among both original and persistent virus clones with different degrees of plating efficiency at 40°/34° C. RNA fingerprinting showed only minor changes in persistent rubella virus.
This study evaluated the influence of different vascular reconstruction kernels on the image quality of CT angiographies of the lower extremity runoff using a 1st-generation photon-counting-detector CT (PCD-CT) compared with dose-matched examinations on a 3rd-generation energy-integrating-detector CT (EID-CT). Inducing continuous extracorporeal perfusion in a human cadaveric model, we performed CT angiographies of eight upper leg arterial runoffs with radiation dose-equivalent 120 kVp acquisition protocols (CTDIvol 5 mGy). Reconstructions were executed with different vascular kernels, matching the individual modulation transfer functions between scanners. Signal-to-noise-ratios (SNR) and contrast-to-noise-ratios (CNR) were computed to assess objective image quality. Six radiologists evaluated image quality subjectively using a forced-choice pairwise comparison tool. Interrater agreement was determined by calculating Kendall’s concordance coefficient (W). The intraluminal attenuation of PCD-CT images was significantly higher than of EID-CT (414.7 ± 27.3 HU vs. 329.3 ± 24.5 HU; p < 0.001). Using comparable kernels, image noise with PCD-CT was significantly lower than with EID-CT (p ≤ 0.044). Correspondingly, SNR and CNR were approximately twofold higher for PCD-CT (p < 0.001). Increasing the spatial frequency for PCD-CT reconstructions by one level resulted in similar metrics compared to EID-CT (CNRfat; EID-CT Bv49: 21.7 ± 3.7 versus PCD-CT Bv60: 21.4 ± 3.5). Overall image quality of PCD-CTA achieved ratings superior to EID-CTA irrespective of the used reconstruction kernels (best: PCD-CT Bv60; worst: EID-CT Bv40; p < 0.001). Interrater agreement was good (W = 0.78). Concluding, PCD-CT offers superior intraluminal attenuation, SNR, and CNR compared to EID-CT in angiographies of the upper leg arterial runoff. Combined with improved subjective image quality, PCD-CT facilitates the use of sharper convolution kernels and ultimately bears the potential of improved vascular structure assessability.
Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) and characterized by the infiltration of immune cells, demyelination and axonal loss. Loss of axons and nerve fiber pathology are widely accepted as correlates of neurological disability. Hence, it is surprising that the development of neuroprotective therapies has been neglected for a long time. A reason for this could be the diversity of the underlying mechanisms, complex changes in nerve fiber pathology and the absence of biomarkers and tools to quantify neuroregenerative processes. Present therapeutic strategies are aimed at modulating or suppressing the immune response, but do not primarily attenuate axonal pathology. Yet, target-oriented neuroprotective strategies are essential for the treatment of MS, especially as severe damage of nerve fibers mostly occurs in the course of disease progression and cannot be impeded by immune modulatory drugs. This review shall depict the need for neuroprotective strategies and elucidate difficulties and opportunities.
Herzkreislauferkrankungen sind weit verbreitet und nicht nur eine große Belastung für die Betroffenen, sondern auch für das Gesundheitssystem. Die Folgen von Herzkreislauferkrankungen wie z.B. Myokardinfarkt und koronare Herzkrankheit stellen weltweit die häufigste Todesursache dar. Prävention, frühzeitige Erkennung und konsequente Behandlung sind daher von großer Bedeutung.
Um das Verständnis für die Pathophysiologie zu fördern und ferner Therapieansätze ausfindig zu machen, ist es notwendig, nicht nur die Herzmuskelzellen im Blick zu haben, sondern auch die Komponenten des Herzmuskelstromas, die deren Funktion beeinflussen können.
Das Verständnis und die Rekonstruktion des kardialen Gewebes auf ultrastruktureller Ebene, sowie die Charakterisierung und Wechselwirkungen der verschiedenen Zellen des Herzens haben deshalb das Interesse vieler Forschergruppen geweckt.
Das Ziel dieser Arbeit war die detaillierte ultrastrukturelle Analyse kardialer Perizyten, Endothelzellen sowie Kapillarwand-assoziierter Zellen und deren Kontakte im Arbeitsmyokard der Maus mittels verschiedener elektronenmikroskopischer Methoden.
Zu Beginn der Arbeit wurde die transmissionselektronenmikroskopische Probenaufbereitung optimiert und ein modifiziertes Protokoll zur hervorragenden Kontrastierung der biologischen Membranen und zum bestmöglichen Erhalt der Ultrastruktur etabliert. Die optimierte Probenaufbereitung bot dann die ideale Grundlage für die Generierung elektronenmikroskopischer Datensätze mittels serieller Block-Face Rasterelektronenmikroskopie (SBF-SEM) und anschließender Erzeugung dreidimensionaler Modelle der Mikrovaskulatur des Arbeitsmyokards der Maus.
Die detaillierte ultrastrukturelle Analyse in drei Dimensionen offenbart neue morphologische Merkmale der kardialen Mikrovaskulatur und zeigt, dass die kardialen Perizyten vereinzelt Fortsätze abgeben, die mit den Endothelzellen assoziiert sind. Dadurch entsteht nicht nur eine perizytäre-endotheliale Einheit, die von derselben Basallamina umschlossen wird. Die Rekonstruktion zeigt ebenfalls, dass die Kapillarwand-assoziierten Zellen sehr groß und weit verzweigt sind und nicht von der die Perizyten und Endothelzellen umgebenden Basallamina umschlossen werden. Sie stehen an vereinzelten Stellen in direktem Kontakt mit den Endothelzellen.
Immunelektronenmikroskopische Analysen zeigen, dass die Kapillarwand-assoziierten Zellen sowohl CD34-positiv als auch CD44-positiv sind.
Größer angelegte Studien zur weiteren dreidimensionalen Analyse z.B. in der Intima einer Arteriole könnten zur weiteren Charakterisierung der Perizyten und der Kapillarwand-assoziierten Zellen beitragen und sogar eine Einteilung möglich machen.
Eine Beteiligung von Perizyten im Rahmen des kardialen Remodeling nach einem Myokardinfarkt wurde bereits nachgewiesen. Außerdem spielen die Membranproteine CD34 und CD44 eine wichtige Rolle in der Hämatopoese und auch der Angiogenese.
In Zukunft könnten sich auch daraus interessante neue Ansätze für gezielte Therapien nach einem Myokardinfarkt ergeben.
Eine Liste der 205 Arten und 1561 Unterarten der Gattung Hieracium s. l. , die in Deutschland, aufgeschlüsselt nach Bundesländern vorkommen, wird vorgestellt. Da die meisten infraspezifischen Namen unter Hieracium publiziert wurden und um die Zahl der invaliden Namen unter Pilosella in der Liste zu minimieren, wird auf eine Aufteilung in Hieracium und Pilosella verzichtet. Durch Farbmarkierungen wird gekennzeichnet, welche Unterart ursprünglich aus einem Bundesland beschrieben wurde bzw. ob ein Syntypus aus einem Bundesland stammt.
Nach bisheriger Kenntnis sind aus den Alpen Vorkommen von fünf Taraxacum-Kleinarten der Sektion Borealia in Frankreich, der Schweiz, Österreich, Italien und Slowenien bekannt: Taraxacum gallicum, Taraxacum handelii, T. kraettlii, T. mazzettii und T. melzerianum. Zwischen 2004 und 2014 wurden diese Vorkommen und weitere potentielle Wuchsorte aufgesucht. Durch detaillierte Untersuchung der Vorkommen vor Ort sowie zahlreicher Belege aus mehreren europäischen Herbarien können nun Merkmale ergänzt, präzisiert und einige Fehler, Unklarheiten in den Originalbeschreibungen korrigiert und Lücken ergänzt werden. Zahlreiche Fotos und Zeichnungen sowie ein neugefasster Schlüssel sollen den Zugang zur Sektion Borealia erleichtern. Mit Taraxacum cimae-gallinae vom Hühnerspiel bei Sterzing (Italien, Südtirol) wird eine neue Art der Sektion Borealia beschrieben. Die Wuchsorte der Borealia-Arten in der alpinen Stufe sind überwiegend Schotterböden auf windgefegten Graten und Gipfelverebnungen. Diese sind derzeit sowohl durch den Ski-Tourismus als auch durch die Klimaerwärmung gefährdet.
In patients with low-risk breast cancer, intraoperative radiotherapy (IORT) during breast-conserving surgery is a novel and convenient treatment option for delivering a single high dose of irradiation directly to the tumour bed. However, edema and fibrosis can develop after surgery and radiotherapy, which can subsequently impair quality of life. TGF-β is a strong inducer of the extracellular matrix component hyaluronan (HA). TGF-β expression and HA metabolism can be modulated by irradiation experimentally, and are involved in edema and fibrosis. We therefore hypothesized that IORT may regulate these factors.Wound fluid (WF) draining from breast lumpectomy sites was collected and levels of TGF-β1 and HA were determined by ELISA. Proliferation and marker expression was analyzed in primary lymphatic endothelial cells (LECs) treated with recombinant TGF-β or WF. Our results show that IORT does not change TGF-β1 or HA levels in wound fluid draining from breast lumpectomy sites, and does not lead to accumulation of sHA oligosaccharides. Nevertheless, concentrations of TGF-β1 were high in WF from patients regardless of IORT, at concentrations well above those associated with fibrosis and the suppression of LEC identity. Consistently, we found that TGF-β in WF is active and inhibits LEC proliferation. Furthermore, all three TGF-β isoforms inhibited LEC proliferation and suppressed LEC marker expression at pathophysiologically relevant concentrations.
Given that TGF-β contributes to edema and plays a role in the regulation of LEC identity, we suggest that inhibition of TGF-β directly after surgery might prevent the development of side effects such as edema and fibrosis.
Ischemic insults to the heart and brain, i.e., myocardial and cerebral infarction, respectively, are amongst the leading causes of death worldwide. While there are therapeutic options to allow reperfusion of ischemic myocardial and brain tissue by reopening obstructed vessels, mitigating primary tissue damage, post-infarction inflammation and tissue remodeling can lead to secondary tissue damage. Similarly, ischemia in retinal tissue is the driving force in the progression of neovascular eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), which eventually lead to functional blindness, if left untreated. Intriguingly, the easily observable retinal blood vessels can be used as a window to the heart and brain to allow judgement of microvascular damages in diseases such as diabetes or hypertension. The complex neuronal and endocrine interactions between heart, retina and brain have also been appreciated in myocardial infarction, ischemic stroke, and retinal diseases. To describe the intimate relationship between the individual tissues, we use the terms heart-brain and brain-retina axis in this review and focus on the role of transforming growth factor β (TGFβ) and neurotrophins in regulation of these axes under physiologic and pathologic conditions. Moreover, we particularly discuss their roles in inflammation and repair following ischemic/neovascular insults. As there is evidence that TGFβ signaling has the potential to regulate expression of neurotrophins, it is tempting to speculate, and is discussed here, that cross-talk between TGFβ and neurotrophin signaling protects cells from harmful and/or damaging events in the heart, retina, and brain.
The Best for the Most Important: Maintaining a Pristine Proteome in Stem and Progenitor Cells
(2019)
Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.
B cells have only recently begun to attract attention in the immunopathology of multiple sclerosis (MS). Suitable markers for the prediction of treatment success with immunomodulatory drugs are still missing. Here we evaluated the B cell response to brain antigens in n = 34 relapsing-remitting MS (RRMS) patients treated with glatiramer acetate (GA) using the enzyme-linked immunospot technique (ELISPOT). Our data demonstrate that patients can be subdivided into responders that show brain-specific B cell reactivity in the blood and patients without this reactivity. Only in patients that classified as B cell responders, there was a significant positive correlation between treatment duration and the time since last relapse in our study. This correlation was GA-specific because it was absent in a control group that consisted of interferon-\(\beta\) (IFN-\(\beta\))-treated RRMS patients (n = 23). These data suggest that GA has an effect on brain-reactive B cells in a subset of patients and that only this subset benefits from treatment. The detection of brain-reactive B cells is likely to be a suitable tool to identify drug responders.
There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.
Besides external characteristics and reading a piece of DNA (barcode), the DNA weight per nucleus (genome size) via flow cytometry is a key value to detect species and hybrids and determine ploidy. In addition, the DNA weight appears to be related to various properties, such as the size of the cell and the nucleus, the duration of mitosis and meiosis and the generation time. Sometimes it is even possible to distinguish between groups or sections, which can lead to new classification of the genera. The variation in DNA weight is also useful to analyze biodiversity, genome evolution and relationships between related taxa. Moreover, it is important to know how large a genome is before one determines the base sequence of the DNA of a plant. Flow cytometry is also important for understanding fundamental processes in plants such as growth and development and recognizing chimeras. In the literature, DNA weight measurements are usually limited to one genus and often only locally (Siljak et al. 2010; Bai et al. 2012). In this study, however, it was decided to investigate all vascular plants from one country. This can also contribute to the protection of rare plants. This study is the first flora in the world whose weight of DNA per nucleus and peak patterns has been determined. More than 6400 plants, representing more than 2350 (sub)species (more than 90%) have been collected, thanks to the help of almost 100 volunteers of Floristisch Onderzoek Nederland (Floron). Multiple specimens of many species have therefore been measured, preferably from different populations, in some cases more than fifty. For 1370 species, these values were not previously published. Moreover, a good number of the remaining 45% are new for The Netherlands. In principle, each species has a fixed weight of DNA per nucleus. It has also been found that, especially between the genera, there are strong differences in the number of peaks that determine the DNA weight, from one to five peaks. This indicates that in a plant or organ there are sometimes nuclei with multiples of its standard DNA weight (multiple ploidy levels). It is impossible to show graphs of more than 2350 species. Therefore, we have chosen to show the peak pattern in a new way in a short formula. Within most genera there are clear differences in the DNA weights per nucleus between the species, in some other genera the DNA weight is hardly variable. Based on about twenty genera that were previously measured completely in most cases (‘t Hart et al. 2003: Veldkamp and Zonneveld 2011; Soes et al. 2012; Dirkse et al. 2014, 2015; Verloove et al. 2017; Zonneveld [et al.] 2000−2018), it can be noted that even if all species of a genus have the same number of chromosomes, there can still be a difference of up to three times in the weight of the DNA. Therefore, a twice larger DNA weight does not have to indicate four sets of chromosomes. Finally, this research has also found clues to examine further the current taxonomy of a number of species or genera.
The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium
(2012)
The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine \((Arfrp1^{vil−/−})\) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. \(Arfrp1^{vil−/−}\) enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the \(Arfrp1^{vil−/−}\) epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylomicrons and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.
Diabetes mellitus is a common disease affecting more than 537 million adults worldwide. The microvascular complications that occur during the course of the disease are widespread and affect a variety of organ systems in the body. Diabetic retinopathy is one of the most common long-term complications, which include, amongst others, endothelial dysfunction, and thus, alterations in the blood-retinal barrier (BRB). This particularly restrictive physiological barrier is important for maintaining the neuroretina as a privileged site in the body by controlling the inflow and outflow of fluid, nutrients, metabolic end products, ions, and proteins. In addition, people with diabetic retinopathy (DR) have been shown to be at increased risk for systemic vascular complications, including subclinical and clinical stroke, coronary heart disease, heart failure, and nephropathy. DR is, therefore, considered an independent predictor of heart failure. In the present review, the effects of diabetes on the retina, heart, and kidneys are described. In addition, a putative common microRNA signature in diabetic retinopathy, nephropathy, and heart failure is discussed, which may be used in the future as a biomarker to better monitor disease progression. Finally, the use of miRNA, targeted neurotrophin delivery, and nanoparticles as novel therapeutic strategies is highlighted.
The Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin superfamily. CEACAM1 was shown to be a prognostic marker in patients suffering from cancer. In this review, we summarize pre-clinical and clinical evidence linking CEACAM1 to tumorigenicity and cancer progression. Furthermore, we discuss potential CEACAM1-based mechanisms that may affect cancer biology.
Background
The key for successful delivery in minimally-invasive hip replacement lies in the exact knowledge about the surgical anatomy. The minimally-invasive direct anterior approach to the hip joint makes it necessary to clearly identify the tensor fasciae latae muscle in order to enter the Hueter interval without damaging the lateral femoral cutaneous nerve. However, due to the inherently restricted overview in minimally-invasive surgery, this can be difficult even for experienced surgeons.
Methods and Surgical Technique
In this technical note, we demonstrate for the first time how to use the tensor fasciae latae perforator as anatomical landmark to reliably identify the tensor fasciae latae muscle in orthopaedic surgery. Such perforators are used for flaps in plastic surgery as they are constant and can be found at the lateral third of the tensor fasciae latae muscle in a direct line from the anterior superior iliac spine.
Conclusion
As demonstrated in this article, a simple knowledge transfer between surgical disciplines can minimize the complication rate associated with minimally-invasive hip replacement.
Background
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.
Methods
Twenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.
Results
B6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.
Conclusions
Our results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.
For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter “taste” substances are most probably initiated by tracheal brush cells (BC). Our single‐cell RNA‐seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca\(^{2+}\)]\(_{i}\) in BC and subsequent ACh‐release. ACh‐release is regulated in an autocrine manner. While the muscarinic ACh‐receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca\(^{2+}\)]\(_{i}\) in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5‐ and M3R‐mediated. We show that ACh‐release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
Operative treatment of ruptured pubic symphysis by plating is often accompanied by complications. Trans-obturator cable fixation might be a more reliable technique; however, have not yet been tested for stabilization of ruptured pubic symphysis. This study compares symphyseal trans-obturator cable fixation versus plating through biomechanical testing and evaluates safety in a cadaver experiment. APC type II injuries were generated in synthetic pelvic models and subsequently separated into three different groups. The anterior pelvic ring was fixed using a four-hole steel plate in Group A, a stainless steel cable in Group B, and a titan band in Group C. Biomechanical testing was conducted by a single-leg-stance model using a material testing machine under physiological load levels. A cadaver study was carried out to analyze the trans-obturator surgical approach. Peak-to-peak displacement, total displacement, plastic deformation and stiffness revealed a tendency for higher stability for trans-obturator cable/band fixation but no statistical difference to plating was detected. The cadaver study revealed a safe zone for cable passage with sufficient distance to the obturator canal. Trans-obturator cable fixation has the potential to become an alternative for symphyseal fixation with less complications.
Transcriptional and distributional profiling of microglia in retinal angiomatous proliferation
(2022)
Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.
Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.
Transportrelevante Substratinteraktionen des organischen Kationentransporters 1 der Ratte (rOCT1)
(2011)
Im Rahmen der vorliegenden Arbeit wurde das mechanistische Funktionsprinzip des Rat Organic Cation Transporter 1, stellvertretend für die Gruppe der organischen Kationentransporter, im Hinblick auf die Beteiligung einzelner Aminosäuren der mutmaßlichen Bindungstasche am Transportprozess (Tyr222, Asp475) untersucht. Hierbei stand insbesondere die Frage nach einer gleichzeitigen oder sequentiellen Interaktion der o. g. Aminosäuren mit dem jeweils gewählten Transportliganden im Vordergrund. Bei Mutation der untersuchten Interaktionsstellen (Einzelmutanten Y222F, D475E, Doppelmutante Y222F/D475E) konnten Km-Änderungen für die zelluläre Aufnahme von MPP und TEA sowie IC50-Änderungen für die Hemmung des MPP-Transportes durch TEA bzw. TBuA im Xenopus-laevis-Oozytenmodell mittels radioaktiver Aufnahmemessungen erzielt werden. Trotz eines signifikant additiven Effektes der Doppelmutation auf die TEA-Affinität des Transporters im Vergleich zu den Einzelmutanten erschien ein sequentieller Transportmechanismus aufgrund der nicht additiven IC50(TEA)-Verminderung für den MPP-Transport und der Sicherung der Lokalisation von Tyr222 und Asp475 auf unterschiedlichen Seiten der Plasmamembran durch TBuA-Inhibitionsversuche der MPP-Aufnahme wahrscheinlich. Gestützt wurden diese Ergebnisse durch die analoge Modellierung dreidimensionaler Darstellungen des doppelmutierten rOCT1(Y222F/D475E) anhand bereits kristallographisch vermessener Prokaryotentransporter. Diese Modelle bestätigten eine interaktionsrelevante räumliche Position der modifizierten Aminosäurereste innerhalb der Bindungstasche, welche eine metachrone Substrat- bzw. Inhibitorbindung nahelegte. Demnach interagiert in Kongruenz beider Untersuchungsansätze zunächst Tyr222 auf extrazellulärer Seite mit dem Liganden, während der intramembranären Konformationsänderung des Transporters erfolgt dann eine Transposition des Substrates respektive Inhibitors auf Asp475 auf der Zytosolseite.
In der vorliegenden Arbeit wird ein neues Teilareal von T. micranthum mit zahlreichen Vorkommen an den Nordseedeichen von Schleswig-Holstein zwischen der Elbeästuar und der Insel Nordstrand mit Schwerpunkt auf der Halbinsel Eiderstedt mitgeteilt, das geographisch zwischen dem Vorkommen in den Niederlanden und dem Ostsee-Areal in Dänemark vermittelt. Es handelt sich um die einzigen weitgehend naturnahen Wuchsorte der Art in Deutschland. Die anderen beiden aktuellen deutschen Vorkommen befinden sich auf Friedhöfen in Nordrhein-Westfalen. T. micranthum wächst bevorzugt an den steilen und artenreicheren Innenböschungen der Seedeiche, deren Vegetation durch intensive Schafbeweidung und Trittspuren kurz und lückig gehalten wird. Die Beweidung bewirkt eine signifikante Größenreduktion (Miniaturisierung) verschiedener Pflanzenteile. Widersprüchliche Angaben zu bestimmungskritischen Merkmalen werden durch morphometrische Untersuchungen überprüft. Unter anderem beträgt die Länge der Blütenstiele 0,6–1,1 mm (im Mittel 0,8 mm) und die Blüten mit Kelch sind deutlich unter 3 mm lang (im Mittel 2,4 mm). Die Zahl der Blüten der Infloreszenz beträgt (1)2–6(8). Eine graphische Darstellung soll bei Artbestimmung und Auffinden neuer Wuchsorte behilflich sein.
Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1 000 nm in diameter), which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter), derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM)-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis.
Ultra-high field cardiac MRI in large animals and humans for translational cardiovascular research
(2023)
A key step in translational cardiovascular research is the use of large animal models to better understand normal and abnormal physiology, to test drugs or interventions, or to perform studies which would be considered unethical in human subjects. Ultrahigh field magnetic resonance imaging (UHF-MRI) at 7 T field strength is becoming increasingly available for imaging of the heart and, when compared to clinically established field strengths, promises better image quality and image information content, more precise functional analysis, potentially new image contrasts, and as all in-vivo imaging techniques, a reduction of the number of animals per study because of the possibility to scan every animal repeatedly. We present here a solution to the dual use problem of whole-body UHF-MRI systems, which are typically installed in clinical environments, to both UHF-MRI in large animals and humans. Moreover, we provide evidence that in such a research infrastructure UHF-MRI, and ideally combined with a standard small-bore UHF-MRI system, can contribute to a variety of spatial scales in translational cardiovascular research: from cardiac organoids, Zebra fish and rodent hearts to large animal models such as pigs and humans. We present pilot data from serial CINE, late gadolinium enhancement, and susceptibility weighted UHF-MRI in a myocardial infarction model over eight weeks. In 14 pigs which were delivered from a breeding facility in a national SARS-CoV-2 hotspot, we found no infection in the incoming pigs. Human scanning using CINE and phase contrast flow measurements provided good image quality of the left and right ventricle. Agreement of functional analysis between CINE and phase contrast MRI was excellent. MRI in arrested hearts or excised vascular tissue for MRI-based histologic imaging, structural imaging of myofiber and vascular smooth muscle cell architecture using high-resolution diffusion tensor imaging, and UHF-MRI for monitoring free radicals as a surrogate for MRI of reactive oxygen species in studies of oxidative stress are demonstrated. We conclude that UHF-MRI has the potential to become an important precision imaging modality in translational cardiovascular research.
Ultra-high-resolution photon-counting detector CT arthrography of the ankle: a feasibility study
(2023)
This study was designed to investigate the image quality of ultra-high-resolution ankle arthrography employing a photon-counting detector CT. Bilateral arthrograms were acquired in four cadaveric specimens with full-dose (10 mGy) and low-dose (3 mGy) scan protocols. Three convolution kernels with different spatial frequencies were utilized for image reconstruction (ρ\(_{50}\); Br98: 39.0, Br84: 22.6, Br76: 16.5 lp/cm). Seven radiologists subjectively assessed the image quality regarding the depiction of bone, hyaline cartilage, and ligaments. An additional quantitative assessment comprised the measurement of noise and the computation of contrast-to-noise ratios (CNR). While an optimal depiction of bone tissue was achieved with the ultra-sharp Br98 kernel (S ≤ 0.043), the visualization of cartilage improved with lower modulation transfer functions at each dose level (p ≤ 0.014). The interrater reliability ranged from good to excellent for all assessed tissues (intraclass correlation coefficient ≥ 0.805). The noise levels in subcutaneous fat decreased with reduced spatial frequency (p < 0.001). Notably, the low-dose Br76 matched the CNR of the full-dose Br84 (p 0.999) and superseded Br98 (p < 0.001) in all tissues. Based on the reported results, a photon-counting detector CT arthrography of the ankle with an ultra-high-resolution collimation offers stellar image quality and tissue assessability, improving the evaluation of miniscule anatomical structures. While bone depiction was superior in combination with an ultra-sharp convolution kernel, soft tissue evaluation benefited from employing a lower spatial frequency.
In this study, the impact of reconstruction sharpness on the visualization of the appendicular skeleton in ultrahigh-resolution (UHR) photon-counting detector (PCD) CT was investigated. Sixteen cadaveric extremities (eight fractured) were examined with a standardized 120 kVp scan protocol (CTDI\(_{vol}\) 10 mGy). Images were reconstructed with the sharpest non-UHR kernel (Br76) and all available UHR kernels (Br80 to Br96). Seven radiologists evaluated image quality and fracture assessability. Interrater agreement was assessed with the intraclass correlation coefficient. For quantitative comparisons, signal-to-noise-ratios (SNRs) were calculated. Subjective image quality was best for Br84 (median 1, interquartile range 1–3; p ≤ 0.003). Regarding fracture assessability, no significant difference was ascertained between Br76, Br80 and Br84 (p > 0.999), with inferior ratings for all sharper kernels (p < 0.001). Interrater agreement for image quality (0.795, 0.732–0.848; p < 0.001) and fracture assessability (0.880; 0.842–0.911; p < 0.001) was good. SNR was highest for Br76 (3.4, 3.0–3.9) with no significant difference to Br80 and Br84 (p > 0.999). Br76 and Br80 produced higher SNRs than all kernels sharper than Br84 (p ≤ 0.026). In conclusion, PCD-CT reconstructions with a moderate UHR kernel offer superior image quality for visualizing the appendicular skeleton. Fracture assessability benefits from sharp non-UHR and moderate UHR kernels, while ultra-sharp reconstructions incur augmented image noise.
Extracellular vesicle (EV)-mediated intercellular communication through exosomes, microvesicles (MVs) and apoptotic bodies has been shown to be implicated in various physiological as well as pathological processes such as the development and progression of atherosclerosis. While the cellular machinery controlling EV formation and composition has been studied extensively, little is known about the underlying morphological processes. This study focuses on a detailed ultrastructural analysis of the different steps of EV formation and release in Myocardial Endothelial (MyEnd) and Aortic Endothelial (AoEnd) cells cultured under serum starvation and inflammatory stimulation with TNF-α. Detailed morphological analyses were conducted applying and comparing different high- resolution light and electron microscopic methods. In this study, we could depict all steps of MV biogenesis named in literature. However, during the study of exosome biogenesis, we discovered a yet undescribed process: Instead of a direct fusion with the plasma membrane, multivesicular bodies were incorporated into a new distinct cellular compartment bound by fenestrated endothelium first. This may present a novel step in exosome biogenesis and warrants further study. Regarding the conditions of cell cultivation, we observed that the commonly used serum starvation causes MyEnd cells, but not AoEnd cells, to enter apoptosis after 48 hours. When preparing functional EV studies, we therefore recommend assessing the morphological condition of the serum-starved cells at different cultivation points first. When evaluating MV production, a statistical analysis showed that the more time AoEnd cells spent in cultivation under serum starvation, the higher the percentage of MV producing cells. However, additional TNF-α stimulation induced a significantly higher MV production than serum starvation alone. Lastly, our results show that TNF-α stimulation of AoEnd cells in vitro leads to the upregulation of CD44, an adhesion molecule critical in the early stages of atherosclerosis. CD44 was then depicted on the surface of generated MVs and exosomes. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells. This could be a novel mechanism by which MVs contribute to the development and progression of atherosclerotic disease and should be clarified by further studies.
Die Tumorangiogenese ist ein Prozess, der zur Ausbildung eines tumoreigenen Gefäßnetzwerks führt und kritisch ist für die Progression des Tumorwachstums, sowie für dessen Malignisierung und Metastasierung. Zytokine wie VEGF und PDGF steuern angiogene Prozesse. Die resultierende Tumorvaskulatur ist jedoch dysfunktional und unterscheidet sich in Struktur und Funktion stark von normalen Gefäßen. Die antiangiogene Therapie richtet sich gegen die Tumorvaskulatur indem Angiogenese-induzierende Signalwege inhibiert werden. Es existieren zahlreiche therapeutische Ansätze, zu denen u.a. Anti-VEGF- Antikörper und Rezeptortyrosinkinaseinhibitoren zählen. Ziel der antiangiogenen Therapie ist es, die Ausbildung neuer Blutgefäße im Tumor zu stoppen sowie existierende unreife Blutgefäße zu zerstören. Das Konzept der Gefäßnormalisierung beschreibt im Rahmen der antiangiogenen Therapie Prozesse, die zu einer transienten Verbesserung dieser defekten Tumorvaskulatur und zu ihrer tendenziellen Angleichung an Struktur und Funktion von normalen Gefäßen führen sollen.
In dieser Studie wurden Veränderungen von Gefäßparametern in murinen AT3- Mammakarzinomen und murinen Lewis-lung-Karzinomen miteinander verglichen, die entweder (a) mit mG6-31, einem monoklonalen Anti-VEGF- Antikörper, (b) mit Axitinib, einem niedermolekularen VEGF-R-/PDGF-R- Tyrosinkinaseinhibitor antiangiogen behandelt oder (c) nicht behandelt wurden. Ziel war es dabei, Aussagen über die antiangiogene Wirksamkeit sowie die Gefäß- normalisierende Effektivität der o.g. Antiangiogenetika zu treffen. In einer bereits abgeschlossenen Forschungsarbeit von Ascheid (vgl. Absatz 7.2) wurden mit dem gleichen Experimentalaufbau wie zuvor beschrieben ebenfalls murine Tumoren hinsichtlich makroskopischer Gefäßstruktur und -organisation untersucht. Dabei wurde aufgezeigt, dass Gefäß-normalisierende Prozesse durch o.g. Angiogenetika in geringem Umfang stattfanden. Die durchgeführte Studie zielte darauf ab, die bereits erfassten Resultate zu komplettieren und somit eine abschließende Aussage über das Auftreten von Gefäßnormalisierung zu ermöglichen.
88
In den mG6-31-/Axitinib-/unbehandelten AT3-/LLC-Tumorschnitten wurden die Parameter Gefäßdichte, Apoptoserate, Proliferationsrate, Perizytenbesatz, Intaktheit der vaskulären Basalmembran und endotheliale Expression von TRPC6-Kanälen immunhistochemisch bzw. mittels Immunfluoreszenz detektiert, mikroskopisch aufgenommen und quantifiziert.
Diese Arbeit zeigt, dass Axitinib deutliche antiangiogene Effekte in der Tumorvaskulatur hervorruft, mG6-31 hingegen wirkt schwächer antiangiogen. Im Unterschied zu den Ergebnissen aus Ascheids Arbeit (Ascheid, 2018) konnten- Effekte auf der Ebene der individuellen Blutgefäße nachgewiesen werden, die in der Literatur als Anzeichen für eine Gefäßnormalisierung beschrieben werden. Wiederum waren diese Effekte unter Axitinib stärker ausgeprägt als unter mG6- 31-Behandlung. Die Resultate beider Forschungsarbeiten zusammengefasst betrachtet, kann man feststellen, dass die Zusammenfassung der gefäßverändernden Effekte, die antiangiogene Wirkstoffe hervorrufen, unter dem Begriff „Normalisierung“ in Frage gestellt werden sollte.