Oxidative Stress and Modification of Renal Vascular Permeability Are Associated with Acute Kidney Injury during P. berghei ANKA Infection

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.

Urinary MCP-1 and RBP: Independent predictors of renal outcome in macroalbuminuric diabetic nephropathy

Background: Albuminuria has been considered a sine qua non condition for the diagnosis of diabetic nephropathy (DN) and has been widely used as a surrogate outcome of chronic kidney disease (CKD). However, recent data suggest that albuminuria may fail as a biomarker in a subset of patients, and the search for novel markers is intense. Methods: We analyzed the role of urinary RBP and of serum and urinary cytokines (TGF-beta, MCP-1 and VEGF) as predictors of the risk of dialysis. doubling of serum creatinine or death (primary outcome. PO) in 56 type 2 diabetic patients with macroalbuminuric DN. Results: Mean follow-up time was 30.7 +/- 10 months. Urinary RBP and MCP-1 were significantly higher in patients presenting the PO, whereas no difference was shown for TGF-beta or VEGF. In the Cox regression, urinary RBP. MCP-1 and VEGF were positively associated and serum VEGF was inversely related to the risk of the PO. However, after adjustments for creatinine clearance, proteinuria, and blood pressure only urinary RBP (OR 11.6; 95% CI 2.7-49.2, p = 0.001 for log RBP) and urinary MCP-1 (OR 11.0; 95% CI 1.6-76.4, p = 0.02 for log MCP-1) remained as significant independent predictors of the PO. Conclusion: Urinary RBP and MCP-1 are independently related to the risk of CKD progression in patients with macroalbuminuric DN. Whether these biomarkers have a role in the setting of normoalbuminuria and microalbuminuria in DN should be further investigated. (C) 2012 Elsevier Inc. All rights reserved.

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

STIM 1/Orai 1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai 1/STIM 1 (stromal interaction molecule I). In addition, we investigated whether ovariectomy in females affects the activation of the Orai 1/STIM 1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM 1 and Orai1, as shown by the blockade of STIM 1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM I and Orai I. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM I expression. These findings suggest that augmented activation of STIM 1/Orai 1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai 1 pathway, contributing to vascular protection observed in female rats.

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Inhibition of Autonomic Storm by Epidural Anesthesia Does Not Influence Cardiac Inflammatory Response After Brain Death in Rats

Background. After brain death (BD) donors usually experience cardiac dysfunction, which is responsible for a considerable number of unused organs. Causes of this cardiac dysfunction are not fully understood. Some authors argue that autonomic storm with severe hemodynamic instability leads to inflammatory activation and myocardial dysfunction. Objectives. To investigate the hypothesis that thoracic epidural anesthesia blocks autonomic storm and improves graft condition by reducing the inflammatory response. Methods. Twenty-eight male Wistar rats (250-350 g) allocated to four groups received saline or bupivacaine via an epidural catheter at various times in relation to brain-death induction. Brain death was induced by a sudden increase in intracranial pressure by rapid inflation of a ballon catheter in the extradural space. Blood gases, electrolytes, and lactate analyses were performed at time zero, and 3 and 6 hours. Blood leukocytes were counted at 0 and 6 hours. After 6 hours of BD, we performed euthanasia to measure vascular adhesion molecule (VCAM)-1, intracellular adhesion molecule (ICAM)-1, interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, Bcl-2 and caspase-3 on cardiac tissue. Results. Thoracic epidural anesthesia was effective to block the autonomic storm with a significant difference in mean arterial pressure between the untreated (saline) and the bupivacaine group before BD (P < .05). However, no significant difference was observed for the expressions of VCAM-1, ICAM-1, TNF-alpha, IL-1 beta, Bcl-2, and caspase-3 (P > .05). Conclusion. Autonomic storm did not seem to be responsible for the inflammatory changes associated with BD; thoracic epidural anesthesia did not modify the expression of inflammatory mediators although it effectively blocked the autonomic storm.

Effects of environmental complexity on neurogenesis in the hippocampal dentate gyrus

Introduction. Postnatal neurogenesis in the hippocampal dentate gyrus, can be modulated by numerous determinants, such as hormones, transmitters and stress. Among the factors positively interfering with neurogenesis, the complexity of the environment appears to play a particularly striking role. Adult mice reared in an enriched environment produce more neurons and exhibit better performance in hippocampus-specific learning tasks. While the effects of complex environments on hippocampal neurogenesis are well documented, there is a lack of information on the effects of living under socio-sensory deprivation conditions. Due to the immaturity of rats and mice at birth, studies dealing with the effects of environmental enrichment on hippocampal neurogenesis were carried out in adult animals, i.e. during a period of relatively low rate of neurogenesis. The impact of environment is likely to be more dramatic during the first postnatal weeks, because at this time granule cell production is remarkably higher than at later phases of development. The aim of the present research was to clarify whether and to what extent isolated or enriched rearing conditions affect hippocampal neurogenesis during the early postnatal period, a time window characterized by a high rate of precursor proliferation and to elucidate the mechanisms underlying these effects. The experimental model chosen for this research was the guinea pig, a precocious rodent, which, at 4-5 days of age can be independent from maternal care. Experimental design. Animals were assigned to a standard (control), an isolated, or an enriched environment a few days after birth (P5-P6). On P14-P17 animals received one daily bromodeoxyuridine (BrdU) injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. Methods. Brain sections were processed for BrdU immunhistochemistry, to quantify the new born and surviving cells. The phenotype of the surviving cells was examined by means of confocal microscopy and immunofluorescent double-labeling for BrdU and either a marker of neurons (NeuN) or a marker of astrocytes (GFAP). Apoptotic cell death was examined with the TUNEL method. Serial sections were processed for immunohistochemistry for i) vimentin, a marker of radial glial cells, ii) BDNF (brain-derived neurotrofic factor), a neurotrophin involved in neuron proliferation/survival, iii) PSA-NCAM (the polysialylated form of the neural cell adhesion molecule), a molecule associated with neuronal migration. Total granule cell number in the dentate gyrus was evaluated by stereological methods, in Nissl-stained sections. Results. Effects of isolation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of BDNF. Though in absolute terms P45 isolated animals had less surviving cells than controls, they showed no differences in survival rate and phenotype percent distribution compared to controls. Evaluation of the absolute number of surviving cells of each phenotype showed that isolated animals had a reduced number of cells with neuronal phenotype than controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that isolated animals had less granule cells than controls (-32% at P18 and -42% at P45). Effects of enrichment. In P18 enriched animals there was an increase in cell proliferation (+26%) compared to controls and a higher expression of BDNF. Though in both groups there was a decline in the number of BrdU-positive cells by P45, enriched animals had more surviving cells (+63) and a higher survival rate than controls. No differences were found between control and enriched animals in phenotype percent distribution. Evaluation of the absolute number of cells of each phenotype showed that enriched animals had a larger number of cells of each phenotype than controls. Looking at the location of cells of each phenotype we found that enriched animals had more new neurons in the granule cell layer and more astrocytes and cells with undetermined phenotype in the hilus. Enriched animals had a higher expression of PSA-NCAM in the granule cell layer and hilus Vimentin immunohistochemistry showed that in enriched animals radial glia cells were more numerous and had more processes.. Granule cell count revealed that enriched animals had more granule cells than controls (+37% at P18 and +31% at P45). Discussion. Results show that isolation rearing reduces hippocampal cell proliferation but does not affect cell survival, while enriched rearing increases both cell proliferation and cell survival. Changes in the expression of BDNF are likely to contribute to he effects of environment on precursor cell proliferation. The reduction and increase in final number of granule neurons in isolated and enriched animals, respectively, are attributable to the effects of environment on cell proliferation and survival and not to changes in the differentiation program. As radial glia cells play a pivotal role in neuron guidance to the granule cell layer, the reduced number of radial glia cells in isolated animals and the increased number in enriched animals suggests that the size of radial glia population may change dynamically, in order to match changes in neuron production. The high PSA-NCAM expression in enriched animals may concur to favor the survival of the new neurons by facilitating their migration to the granule cell layer. Conclusions. By using a precocious rodent we could demonstrate that isolated/enriched rearing conditions, at a time window during which intense granule cell proliferation takes place, lead to a notable decrease/increase of total granule cell number. The time-course and magnitude of postnatal granule cell production in guinea pigs are more similar to the human and non-human primate condition than in rats and mice. Translation of current data to humans would imply that exposure of children to environments poor/rich of stimuli may have a notably large impact on dentate neurogenesis and, very likely, on hippocampus dependent memory functions.

Untersuchungen zur induzierbaren Expression des Adhäsionsmoleküls E-Selektin und dessen potentieller Bedeutung bei der Tumormetastasierung

In der vorliegenden Arbeit konnte gezeigt werden, dass sowohl die durch TNFalpha-, als auch durch Röntgenstrahlen vermittelte Expression des Adhäsionsmoleküls E-Selektin in Endothelzellen durch kleine GTPasen der Rho-Proteinfamilie reguliert ist. Hemmung dieser kleinen G-Proteine z.B. durch HMG-CoA Reduktase-Inhibitoren (Statine) oder clostridiale Toxine führt zu verminderter Expression von E-Selektin in humanen Endothelzellen (HUVEC; EA.hy-926). Aus den in der Arbeit erhaltenen Ergebnissen kann außerdem die Schlussfolgerung gezogen werden, dass die Regulation der zytokininduzierten E-Selektin-Genexpression von der gamma-Strahlen-vermittelten Expression des E-Selektingens differiert. Für die strahleninduzierte endotheliale E-Selektin-Expression scheint beispielsweise der Transkriptionsfaktor AP-1 als ein weiterer Kontrollfaktor neben NF-kappaB zu fungieren. Des Weiteren konnte in der vorliegenden Arbeit gezeigt werden, dass eine gesteigerte E-Selektin-Proteinexpression mit erhöhter Tumorzelladhäsion und -transmigration an bzw. durch humane Endothelzellen korreliert. Hemmung der TNFalpha- bzw. gamma-Strahlen-induzierten Expression von E-Selektin durch Statine oder Retinsäuren ist ausreichend, sowohl Tumorzelladhäsion als auch Tumorzelldiapedese zu reduzieren. Zusammenfassend lässt sich festhalten, dass das Adhäsionsmolekül E-Selektin eine vielversprechende Zielstruktur ist, über deren kontrollierte Beeinflussung es auch in vivo möglich sein sollte, das Risiko der Metastasierung zu reduzieren. Mit Statinen und Retinsäurederivaten wurden somit Pharmaka identifiziert, die durch Hemmung der Rho-regulierten E-Selektin-Expression der Tumorzellmetastasierung vorbeugen könnten. Um diese Hypothese in zukünftigen tierexperimentelle Studien zu bestätigen, wurde im Rahmen dieser Arbeit mit der Generierung transgener Tiermodelle begonnen.

Molekulare Ursachen und therapeutische Intervention bei der Alzheimer-Demenz

Ein charakteristisches, neuropathologisches Merkmal der Alzheimer-Demenz (AD), der am häufigsten vorkommenden Demenz-Form des Menschen, ist das Auftreten von senilen Plaques im Gehirn der Patienten. Hierbei stellt das neurotoxische A-beta Peptid den Hauptbestandteil dieser Ablagerungen dar. Einen Beitrag zu der pathologisch erhöhten A-beta Generierung liefert das verschobene Expressionsgleichgewicht der um APP-konkurrierenden Proteasen BACE-1 und ADAM10 zu Gunsten der beta-Sekretase BACE-1. In der vorliegenden Dissertation sollten molekulare Mechanismen identifiziert werden, die zu einem pathologisch veränderten Gleichgewicht der APP-Spaltung und somit zum Entstehen und Fortschritt der AD beitragen. Des Weiteren sollten Substanzen identifiziert werden, die durch Beeinflussung der Genexpression einer der beiden Proteasen das physiologische Gleichgewicht der APP-Prozessierung wiederherstellen können und somit therapeutisch einsetzbar sind.rnAnhand eines „Screenings“ von 704 Transkriptionsfaktoren wurden 23 Faktoren erhalten die das Verhältnis ADAM10- pro BACE-1-Promotor Aktivität beeinflussten. Exemplarisch wurden zwei der molekularen Faktoren auf ihren Wirkmechanismus untersucht: Der TF „X box binding protein-1“ (XBP-1), der die so genannte „unfolded protein response“ (UPR) reguliert, erhöhte die Expression von ADAM10 in Zellkultur-Experimenten. Die Menge dieses Faktors war in AD-Patienten im Vergleich zu gesunden, Alters-korrelierten Kontrollen signifikant erniedrigt. Im Gegensatz dazu verminderte der Seneszenz-assoziierte TF „T box 2“ (Tbx2) die Menge an ADAM10 in SH-SY5Y Zellen. Die Expression des Faktors selbst war in post-mortem Kortexgewebe von AD-Patienten erhöht. Zusätzlich zu den TFs konnten in einer Kooperation mit dem Helmholtz Zentrum München drei microRNAs (miRNA 103, 107, 1306) bioinformatisch prädiziert und experimentell validiert werden, die die Expression des humanen ADAM10 reduzierten.rnIm Rahmen dieser Arbeit konnten damit körpereigene Faktoren identifiziert werden, die die Menge an ADAM10 regulieren und folglich potenziell an der Entstehung der gestörten Homöostase der APP-Prozessierung beteiligt sind. Somit ist die AD auch im Hinblick auf eine A-beta-vermittelte Pathologie als multifaktorielle Krankheit zu verstehen, in der verschiedene Regulatoren zur gestörten APP-Prozessierung und somit zur pathologisch gesteigerten A-beta Generierung beitragen können. rnEine pharmakologische Erhöhung der ADAM10 Genexpression würde zu der Freisetzung von neuroprotektivem APPs-alpha und gleichzeitig zu einer reduzierten A-beta Generierung führen. Deshalb war ein weiteres Ziel dieser Arbeit die Evaluierung von Substanzen mit therapeutischem Potenzial im Hinblick auf eine erhöhte ADAM10 Expression. Von 640 FDA-zugelassenen Medikamenten einer Substanz-Bibliothek wurden 23 Substanzen identifiziert, die die Menge an ADAM10 signifikant steigerten während die Expression von BACE-1 und APP unbeeinflusst blieb. In Zusammenarbeit mit dem Institut für Pathologie (Johannes Gutenberg Universität Mainz) wurde ein Zellkultur-basiertes Modell etabliert, um die Permeationsfähigkeit der potenziellen Kandidaten-Substanzen über die Blut-Hirn Schranke (BHS) zu untersuchen. Von den 23 Medikamenten konnten neun im Rahmen des etablierten Modells als BHS-gängig charakterisiert werden. Somit erfüllen diese verbleibenden Medikamente die grundlegenden Anforderungen an ein AD-Therapeutikum. rnADAM10 spaltet neben APP eine Vielzahl anderer Substrate mit unterschiedlichen Funktionen in der Zelle. Zum Beispiel reguliert das Zelladhäsionsmolekül Neuroligin-1 (NL-1), das von ADAM10 prozessiert wird, die synaptische Funktion exzitatorischer Neurone. Aus diesem Grund ist die Abschätzung potenzieller, Therapie-bedingter Nebenwirkungen sehr wichtig. Im Rahmen eines Forschungsaufenthalts an der Universität von Tokio konnte in primären, kortikalen Neuronen der Ratte bei einer Retinoid-induzierten Erhöhung von ADAM10 neben einer vermehrten alpha-sekretorischen APP-Prozessierung auch eine gesteigerte Spaltung von NL-1 beobachtet werden. Dies lässt vermuten, dass bei einer Behandlung mit dem Retinoid Acitretin neben einer vermehrten APP-Spaltung durch ADAM10 auch die Regulation glutamaterger Neurone durch die Spaltung von NL-1 betroffen ist. Anhand eines geeigneten Alzheimer-Tiermodells sollten diese Befunde weiter analysiert werden, um so auf einen sicheren therapeutischen Ansatz bezüglich einer vermehrten ADAM10 Genexpression schließen zu können.rn

Expanding the scope of poly(ethylene glycol)s for bioconjugation to squaric acid-mediated PEGylation and pH-sensitivity

Poly(ethylene glycol) (PEG) is used in a broad range of applications due to its unique combination of properties and is approved use in formulations for body-care products, edibles and medicine. This thesis aims at the synthesis and characterization of novel heterofunctional PEG structures and the establishment of diethyl squarate as a suitable linker for the covalent attachment to proteins. Chapter 1 is an introduction on the properties and applications of PEG as well as the fascinating chemistry of squaric acid derivatives. In Chapter 1.1, the synthesis and properties of PEG are described, and the versatile applications of PEG derivatives in everyday products are emphasized with a focus on PEG-based pharmaceuticals and nonionic surfactants. This chapter is written in German, as it was published in the German Journal Chemie in unserer Zeit. Chapter 1.2 deals with PEGs major drawbacks, its non-biodegradability, which impedes parenteral administration of PEG conjugates with polyethers exceeding the renal excretion limit, although these would improve blood circulation times and passive tumor targeting. This section gives a comprehensive overview of the cleavable groups that have been implemented in the polyether backbone to tackle this issue as well as the synthetic strategies employed to accomplish this task. Chapter 1.3 briefly summarizes the chemical properties of alkyl squarates and the advantages in protein conjugation chemistry that can be taken from its use as a coupling agent. In Chapter 2, the application of diethyl squarate as a coupling agent in the PEGylation of proteins is illustrated. Chapter 2.1 describes the straightforward synthesis and characterization of squaric acid ethyl ester amido PEGs with terminal hydroxyl functions or methoxy groups. The reactivity and selectivity of theses activated PEGs are explored in kinetic studies on the reactions with different lysine and other amino acid derivatives, followed by 1H NMR spectroscopy. Further, the efficient attachment of the novel PEGs to a model protein, i.e., bovine serum albumin (BSA), demonstrates the usefulness of the new linker for the PEGylation with heterofunctional PEGs. In Chapter 2.3 initial studies on the biocompatibility of polyether/BSA conjugates synthesized by the squaric acid mediated PEGylation are presented. No cytotoxic effects on human umbilical vein endothelial cells exposed to various concentrations of the conjugates were observed in a WST-1 assay. A cell adhesion molecule - enzyme immunosorbent assay did not reveal the expression of E-selectin or ICAM-1, cell adhesion molecules involved in inflammation processes. The focus of Chapter 3 lies on the syntheses of novel heterofunctional PEG structures which are suitable candidates for the squaric acid mediated PEGylation and exhibit superior features compared to established PEGs applied in bioconjugation. Chapter 3.1 describes the synthetic route to well-defined, linear heterobifunctional PEGs carrying a single acid-sensitive moiety either at the initiation site or at a tunable position in the polyether backbone. A universal concept for the implementation of acetal moieties into initiators for the anionic ring-opening polymerization (AROP) of epoxides is presented and proven to grant access to the degradable PEG structures aimed at. The hydrolysis of the heterofunctional PEG with the acetal moiety at the initiating site is followed by 1H NMR spectroscopy in deuterium oxide at different pH. In an exploratory study, the same polymer is attached to BSA via the squarate acid coupling and subsequently cleaved from the conjugate under acidic conditions. Furthermore, the concept for the generation of acetal-modified AROP initiators is demonstrated to be suitable for cholesterol, and the respective amphiphilic cholesteryl-PEG is cleaved at lowered pH. In Chapter 3.2, the straightforward synthesis of α-amino ω2-dihydroxyl star-shaped three-arm PEGs is described. To assure a symmetric length of the hydroxyl-terminated PEG arms, a novel AROP initiator is presented, who’s primary and secondary hydroxyl groups are separated by an acetal moiety. Upon polymerization of ethylene oxide for these functionalities and subsequent cleavage of the acid-labile unit no difference in the degree of polymerization is seen for both polyether fragments.

Die Rolle der Oligodendrozyten-Vorläuferzellen im Zentralen Nervensystem

Im Fokus dieser Studie stehen die zu den Gliazellen zählenden OPC, sowie das von diesen exprimierte Typ-1 Membranprotein NG2. Dieses wird auf eine Prozessierung durch α- und γ-Sekretase, in Analogie zu Proteinen wie Notch oder APP, untersucht.rnEine solche Prozessierung ginge mit zusätzlichen intrazellulären Spaltprodukten neben der bekannten Ektodomäne einher. Da OPC mit dem Neuronalen Netzwerk durch synaptische Innervierungen in Verbindung stehen, stellt sich die Frage, ob diese mit der Spaltung von NG2 in Verbindung gebracht werden können. Dazu käme mechanistisch beispielsweise eine aktivitätsabhängige Regulierung der Proteolyse, wie sie jüngst für das neuronale synaptische cell adhesion molecule Neuroligin gezeigt werden konnte, in Frage. Zudem werden eine physiologische Rolle der NG2 Ektodomäne bzw. der möglichen intrazellulären Fragmente untersuchen. Insbesondere potentielle neuromodulatorische Funktionen sind hier von Interesse, da diese die OPC tiefer in das Neuronale Netzwerk integrieren würden. Die Existenz eines NG2 Homologes in D. melanogaster, wirft weiterhin die Frage auf, in wie weit diese Mechanismen in diesem Modellsystem konserviert sind.rnIn Analogie zur Lokalisierung von Markerproteinen an Neuron-Neuron Synapsen in vivo, ergibt sich die Frage ob sich die synaptischen Verbindungen zwischen Neuronen und OPC in ähnlicher Weise darstellen lassen.rnEin Charakteristikum von OPC ist die Teilungsaktivität in sich entwickelnden und adulten Säugern. Zudem gibt es Evidenzen für direkte funktionelle Verknüpfungen zwischen dem NG2 Protein und dem Teilungsmodus der OPC. Deshalb war ein weiteres Ziel mögliche Änderungen in der Zellteilung der OPC, die mit dem NG2 Protein in Verbindung stehen könnten, in NG2 -/- Mäusen zu untersuchen.rn

MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity

Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

Common obesity risk alleles in childhood attention-deficit/hyperactivity disorder

Children with attention-deficit/hyperactivity disorder (ADHD) have a higher rate of obesity than children without ADHD. Obesity risk alleles may overlap with those relevant for ADHD. We examined whether risk alleles for an increased body mass index (BMI) are associated with ADHD and related quantitative traits (inattention and hyperactivity/impulsivity). We screened 32 obesity risk alleles of single nucleotide polymorphisms (SNPs) in a genome-wide association study (GWAS) for ADHD based on 495 patients and 1,300 population-based controls and performed in silico analyses of the SNPs in an ADHD meta-analysis comprising 2,064 trios, 896 independent cases, and 2,455 controls. In the German sample rs206936 in the NUDT3 gene (nudix; nucleoside diphosphate linked moiety X-type motif 3) was associated with ADHD risk (OR: 1.39; P = 3.4 × 10(-4) ; Pcorr  = 0.01). In the meta-analysis data we found rs6497416 in the intronic region of the GPRC5B gene (G protein-coupled receptor, family C, group 5, member B; P = 7.2 × 10(-4) ; Pcorr  = 0.02) as a risk allele for ADHD. GPRC5B belongs to the metabotropic glutamate receptor family, which has been implicated in the etiology of ADHD. In the German sample rs206936 (NUDT3) and rs10938397 in the glucosamine-6-phosphate deaminase 2 gene (GNPDA2) were associated with inattention, whereas markers in the mitogen-activated protein kinase 5 gene (MAP2K5) and in the cell adhesion molecule 2 gene (CADM2) were associated with hyperactivity. In the meta-analysis data, MAP2K5 was associated with inattention, GPRC5B with hyperactivity/impulsivity and inattention and CADM2 with hyperactivity/impulsivity. Our results justify further research on the elucidation of the common genetic background of ADHD and obesity.