RPE-Chorioidea Transplatation bei altersbedingter Makuladegeneration: Untersuchungen zur Plasmaskalpell-gestützten Abtragung von Chorioidea am Schweinebulbus

Das retinale Pigmentepithel (RPE) ist ein einschichtiges, kuboides Epithel zwischen den retinalen Photorezeptoren und der Chorioidea. Bei der altersbedingten Makuladegeneration (AMD) kommt es zu einem Verlust dieser Epithelschicht entweder durch den degenerativen Krankheitsprozess selbst (nicht exsudative AMD) oder im Falle der chirurgischen Behandlung der exsudativen AMD während der operativen Extraktion einer chorioidalen Neovaskularisation (CNV). Es wurden viele Versuche durchgeführt, die Integrität dieses Epithels durch eine RPE- Transplantation wiederherzustellen. Versuche RPE-Zellsuspensionen subretinal zu injizieren scheiterten, da keine Einschichtigkeit des Pigmentepithels hergestellt werden konnte und die Polarisation der injizierten Zellen unklar blieb (Lappas et al. 2004, van Meurs et al. 2004). Auch Experimente mit allogenen RPE-Chorioidea Transplantaten führten nicht zum Erfolg, da es zu einer subretinalen Fibrosereaktion als Zeichen der Transplantatabstoßung kam (Algvere et al. 1994, Algvere et al. 2006). Die beste Lösung, das degenerierte RPE möglichst in seinem natürlichen Aufbau zu ersetzen scheint deshalb ein autologes RPE-Chorioidea Translokat zu sein, welches aus einschichtigem RPE, Bruch-Membran (Basalmembran) und der Chorioidea besteht. Diese Technik wurde erstmals von Peyman (Payman et al. 1991) beschrieben und inzwischen konnte in mehreren Fallserien gezeigt werden, dass dieses Operationsverfahren vergleichsweise komplikationsarm durchführbar ist und postoperativ eine retinale Lichtempfindlichkeit und Fixation auf dem Translokat möglich ist (Treumer et al. 2007, Joussen et al. 2006, Stanga et al. 2002, van Meurs et al. 2003). Jedoch bleibt der postoperative Visus meist auf einem niedrigen Niveau. Das histologische Korrelat für diese schwachen funktionellen Ergebnisse könnte die am Schweineauge in einigen Fällen beobachtete Degeneration der über dem Translokat liegenden neuronalen Netzhautschichten sein. Interessanterweise wurde diese Degeneration auch beobachtet, wenn das Translokat vital war und histologisch Anschluss an die chorioidale Vaskularisation gefunden hatte (MacLaren et al. 2005). Eine Ursache hierfür könnte sein, dass die Chorioideaschicht des Translokates zu dick war. Die postoperative Vaskularisation des RPE-Chorioidea Translokates nimmt einige Wochen in Anspruch. Während dieser Zeit muss das RPE und die neuronale Netzhaut durch Diffusion von Nährstoffen und Sauerstoff aus der tiefliegenden, intakten Chorioidea des Empfängerbettes ernährt werden. Eine chirurgische Ausdünnung der Chorioideaschicht des RPE-Chorioidea- Translokates könnte die nutritive Versorgung des RPE und der neuronalen Netzhaut in dieser kritischen Zeitspanne verbessern und damit möglicherweise einer Degeneration dieser Zellschichten entgegenwirken. Unter dieser Annahme führten Hu et al. autologe „partial thickness“ RPE-Chorioidea Translokationen am Kaninchenauge durch (Hu et al. 2008). Hierbei dünnten sie die Chorioideaschicht bei der Präparation des Translokates intraoperativ mit einem Spatel aus. Sie konnten dadurch die Gesamtdicke des Translokates auf ca. 50-60 μm reduzieren. Histologisch zeigte sich ein vitales RPE und eine intakte neuronale Netzhaut bei 17 von 25 operierten Tieren mit einem Follow-up von 6 Monaten. Immunhistologisch ließ sich die Expression des RPE-Markers CRALBP in den RPE Zellen nachweisen als Zeichen der weiter bestehenden Differenzierung dieser Zellen. Auch die Expression von Rhodopsin konnte in allen dieser 17 Fälle sowohl im RPE als auch in den Photorezeptoren nachgewiesen werden und deutet auf einen gut erhaltenen Metabolismus zwischen den RPE Zellen und der Photorezeptorschicht hin. Die intraoperative Ausdünnung der Chorioideaschicht des Translokates mit einem Spatel ist jedoch vergleichsweise grob und ungenau zu reproduzieren, die mechanische Belastung für das nur wenige Mikrometer dicke Translokat ist stark und Lochbildungen im Translokat sind schwierig zu vermeiden. Daher wurde in dieser Arbeit die kontrollierte Ausdünnung der Chorioideaschicht mit einem Plasmaskalpell untersucht. Dabei wurde festgestellt, dass vor allem mit zwei Parametern die Wirkung des Plasmaskalpells variiert werden kann. Es zeigte sich ein annähernd linearer Zusammenhang zwischen dem erreichten Gewebeabtrag und den Parametern „Abstand der Skalpellspitze“ von der Chorioidea und „Amplitude der angelegten Spannung“. Der erreichte Gewebeabtrag bei Variation des Parameters „Dauer der Plasma-Einwirkung“ zeigte dagegen ein Plateau im Bereich zwischen 4 und 8 Sekunden. Das heißt, dass in dieser Zeitspanne eine längere Einwirkzeit des Plasmas nicht zu einem erhöhten Gewebeabtrag der Chorioideaschicht führt. Dieses Plateau ist über die begrenzte räumliche Ausdehnung des Plasmas je nach angelegter Spannung zu erklären und bietet einen guten Schutz vor einer ungewollt tiefen Abtragung. In den histologischen und elektronenmikroskopischen Präparaten konnte eine scharf begrenzte Ablation ohne thermischen Kollateralschaden des angrenzenden Gewebes aufgezeigt werden. Die Handhabung des Skalpells war schnell zu erlernen. Die Parameter „Amplitude der Spannung“ und „Abstand“ dienten dazu, die Abtragungsstärke während einer Ablation zu variieren und auf die jeweilige Situation und das Transplantat anzupassen. Bei flächigen Abtragungen konnte durch kreisende Bewegung mit der Instrumentenspitze eine sehr gleichmäßige Ausdünnung erreicht werden. Die weiteren Parameter des Gerätes Zeiss PEAK-FC Plasmaskalpell „Wiederholungsrate [Hz]“ und „Pulse pro Stoß [Anzahl der Stöße]“ wurden immer auf den maximalen Wert eingestellt, um eine gleichmäßige Abtragung zu erreichen (maximale Pulse pro Stoß = 20; maximale Wiederholungsrate = 80Hz). Bei niedrigeren Geräteeinstellungen war nur eine sehr geringe und oft nicht kontinuierliche Abtragung zu erzielen. Der Abstand zum Präparat konnte dadurch sehr gut eingeschätzt werden und gut korrigiert werden. Weiter hat sich bewährt, die Nadelspitze leicht umzubiegen und diese parallel statt senkrecht zum Transplantat zu führen. Hierdurch konnte eine gleichmäßigere Flächenabtragung erreicht werden. Wurde die Nadel senkrecht aufgesetzt bestand die Gefahr in bindegewebigen Oberflächenstrukturen der Choriokapillaris hängen zu bleiben und ein Loch in das Transplantat zu schneiden. Das Plasmaskalpell bietet also die Möglichkeit über den Abstand zum Gewebe und über die eingestellte Leistung die Intensität der Ablation gut zu dosieren und damit eine flächige und zur Tiefe hin begrenzte Gewebeabtragung zu erreichen. An der Instrumentenspitze steht eine fiberoptische Lichtquelle zur Verfügung die das Operationsfeld optimal ausleuchtet und die Ablation gut kontrollieren lässt. Einen weiteren Vorteil bietet das Plasmaskalpell dadurch, dass sich das abgetragene Material in der Plasmawolke auflöst und nicht feine Gewebepartikel im Glaskörper fluktuieren und die intraoperativen Sichtverhältnisse verschlechtern. Angesichts der günstigen Ergebnisse der intravitrealen VEGF-Blockade sind komplexe netzhautchirurgische Verfahren zur Behandlung der exsudativen AMD in den Hintergrund getreten. Dennoch kann eine autologe RPE-Chorioidea Translokation in Einzelfällen erwogen werden. So ist diese operative Option beispielsweise bei Patienten, die nicht auf eine anti- VEGF Therapie ansprechen indiziert, so lange die zentrale neurosensorische Netzhaut noch gut erhalten ist. Auch Augen mit frischen Einrissen des retinalen Pigmentepithels oder frische Pigmentepithelunterblutungen, ggf. mit Durchbruch nach subretinal, weisen meist noch eine gut erhaltene neurosensorische Netzhaut auf und können somit von einer autologen RPE- Chorioidea Translokation profitieren. Die in dieser Arbeit durchgeführten Untersuchungen zeigen, dass es möglich ist, die Aderhautschicht eines RPE-Chorioidea Translokates mit einem Plasmaskalpell kontrolliert und sicher auszudünnen. Weitere klinisch experimentelle Fallserien müssen nun zeigen, ob die auf diese Weise modifizierten Translokate durch eine verbesserte nutritive Versorgung in der Frühphase nach der Translokation eine bessere postoperative Funktion aufweisen und diese längerfristig aufrecht erhalten können

Discovery of Sexual Dimorphisms in Metabolic and Genetic Biomarkers

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8 x 10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8 x 10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation

Viewpoints on emergent semantics

Authors include:Philippe Cudr´e-Mauroux, and Karl Aberer (editors), Alia I. Abdelmoty, Tiziana Catarci, Ernesto Damiani, Arantxa Illaramendi, Robert Meersman, Erich J. Neuhold, Christine Parent, Kai-Uwe Sattler, Monica Scannapieco, Stefano Spaccapietra, Peter Spyns, and Guy De Tr´eWe introduce a novel view on how to deal with the problems of semantic interoperability in distributed systems. This view is based on the concept of emergent semantics, which sees both the representation of semantics and the discovery of the proper interpretation of symbols as the result of a self-organizing process performed by distributed agents exchanging symbols and having utilities dependent on the proper interpretation of the symbols. This is a complex systems perspective on the problem of dealing with semantics. We highlight some of the distinctive features of our vision and point out preliminary examples of its applicatio

Effects of smoking and smoking cessation on human serum metabolite profile: results from the KORA cohort study

Background: Metabolomics helps to identify links between environmental exposures and intermediate biomarkers of disturbed pathways. We previously reported variations in phosphatidylcholines in male smokers compared with non-smokers in a cross-sectional pilot study with a small sample size, but knowledge of the reversibility of smoking effects on metabolite profiles is limited. Here, we extend our metabolomics study with a large prospective study including female smokers and quitters. Methods: Using targeted metabolomics approach, we quantified 140 metabolite concentrations for 1,241 fasting serum samples in the population-based Cooperative Health Research in the Region of Augsburg (KORA) human cohort at two time points: baseline survey conducted between 1999 and 2001 and follow-up after seven years. Metabolite profiles were compared among groups of current smokers, former smokers and never smokers, and were further assessed for their reversibility after smoking cessation. Changes in metabolite concentrations from baseline to the follow-up were investigated in a longitudinal analysis comparing current smokers, never smokers and smoking quitters, who were current smokers at baseline but former smokers by the time of follow-up. In addition, we constructed protein-metabolite networks with smoking-related genes and metabolites. Results: We identified 21 smoking-related metabolites in the baseline investigation (18 in men and six in women, with three overlaps) enriched in amino acid and lipid pathways, which were significantly different between current smokers and never smokers. Moreover, 19 out of the 21 metabolites were found to be reversible in former smokers. In the follow-up study, 13 reversible metabolites in men were measured, of which 10 were confirmed to be reversible in male quitters. Protein-metabolite networks are proposed to explain the consistent reversibility of smoking effects on metabolites. Conclusions: We showed that smoking-related changes in human serum metabolites are reversible after smoking cessation, consistent with the known cardiovascular risk reduction. The metabolites identified may serve as potential biomarkers to evaluate the status of smoking cessation and characterize smoking-related diseases

Discovery of Sexual Dimorphisms in Metabolic and Genetic Biomarkers

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8 x 10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8 x 10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation

Human serum metabolic profiles are age dependent

Understanding the complexity of aging is of utmost importance. This can now be addressed by the novel and powerful approach of metabolomics. However, to date, only a few metabolic studies based on large samples are available. Here, we provide novel and specific information on age-related metabolite concentration changes in human homeostasis. We report results from two population-based studies: the KORA F4 study from Germany as a discovery cohort, with 1038 female and 1124 male participants (32–81 years), and the TwinsUK study as replication, with 724 female participants. Targeted metabolomics of fasting serum samples quantified 131 metabolites by FIA-MS/MS. Among these, 71/34 metabolites were significantly associated with age in women/men (BMI adjusted). We further identified a set of 13 independent metabolites in women (with P values ranging from 4.6 × 10−04 to 7.8 × 10−42, αcorr = 0.004). Eleven of these 13 metabolites were replicated in the TwinsUK study, including seven metabolite concentrations that increased with age (C0, C10:1, C12:1, C18:1, SM C16:1, SM C18:1, and PC aa C28:1), while histidine decreased. These results indicate that metabolic profiles are age dependent and might reflect different aging processes, such as incomplete mitochondrial fatty acid oxidation. The use of metabolomics will increase our understanding of aging networks and may lead to discoveries that help enhance healthy aging

Clawed forelimbs allow northern seals to eat like their ancient ancestors

Funding for this project was provided by a Marie Skłodowska-Curie Global Postdoctoral Fellowship (656010/MYSTICETI) to F.G.M, by Marine Scotland to support the wild observations recorded by R.N.H., by an Australian Research Council Future Fellowship FT130100968 to A.R.E., and by an Australian Research Council Linkage Project LP150100403 to A.R.E. and E.M.G.F.Streamlined flippers are often considered the defining feature of seals and sea lions, whose very name ‘pinniped’ comes from the Latin pinna and pedis, meaning ‘fin-footed’. Yet not all pinniped limbs are alike. Whereas otariids (fur seals and sea lions) possess stiff streamlined forelimb flippers, phocine seals (northern true seals) have retained a webbed yet mobile paw bearing sharp claws. Here, we show that captive and wild phocines routinely use these claws to secure prey during processing, enabling seals to tear large fish by stretching them between their teeth and forelimbs. ‘Hold and tear’ processing relies on the primitive forelimb anatomy displayed by phocines, which is also found in the early fossil pinniped Enaliarctos. Phocine forelimb anatomy and behaviour therefore provide a glimpse into how the earliest seals likely fed, and indicate what behaviours may have assisted pinnipeds along their journey from terrestrial to aquatic feeding.Publisher PDFPeer reviewe

Body Fat Free Mass Is Associated with the Serum Metabolite Profile in a Population-Based Study

To characterise the influence of the fat free mass on the metabolite profile in serum samples from participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) S4 study. Analyses were based on metabolite profile from 965 participants of the S4 and 890 weight-stable subjects of its seven-year follow-up study (KORA F4). 190 different serum metabolites were quantified in a targeted approach including amino acids, acylcarnitines, phosphatidylcholines (PCs), sphingomyelins and hexose. Associations between metabolite concentrations and the fat free mass index (FFMI) were analysed using adjusted linear regression models. To draw conclusions on enzymatic reactions, intra-metabolite class ratios were explored. Pairwise relationships among metabolites were investigated and illustrated by means of Gaussian graphical models (GGMs). We found 339 significant associations between FFMI and various metabolites in KORA S4. Among the most prominent associations (p-values 4.75 × 10(-16)-8.95 × 10(-06)) with higher FFMI were increasing concentrations of the branched chained amino acids (BCAAs), ratios of BCAAs to glucogenic amino acids, and carnitine concentrations. For various PCs, a decrease in chain length or in saturation of the fatty acid moieties could be observed with increasing FFMI, as well as an overall shift from acyl-alkyl PCs to diacyl PCs. These findings were reproduced in KORA F4. The established GGMs supported the regression results and provided a comprehensive picture of the relationships between metabolites. In a sub-analysis, most of the discovered associations did not exist in obese subjects in contrast to non-obese subjects, possibly indicating derangements in skeletal muscle metabolism. A set of serum metabolites strongly associated with FFMI was identified and a network explaining the relationships among metabolites was established. These results offer a novel and more complete picture of the FFMI effects on serum metabolites in a data-driven network

Differences between Human Plasma and Serum Metabolite Profiles

BACKGROUND: Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r) of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01). Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10), which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D) patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices. CONCLUSIONS/SIGNIFICANCE: Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is used, either matrix should generate similar results in clinical and biological studies. The higher metabolite concentrations in serum, however, make it possible to provide more sensitive results in biomarker detection