Identifizierung und Charakterisierung von Liganden für Faktor VIII neutralisierende Antikörper

Das Fehlen von funktionellem Blutgerinnungsfaktor VIII (FVIII) in Hämophilie A- (HA-) Patienten wird durch Substitution mit FVIII-Präparaten therapiert. Die wesentlichste gegenwärtige Komplikation der FVIII-Ersatz-Therapie besteht in dem Auftreten von FVIII neutralisierenden Antikörpern (Inhibitoren) gegenüber exogenem FVIII. Diese können mittels verschiedener, kostenintensiver Therapien zur Induktion einer Immuntoleranz (ITI) mit unterschiedlichem Erfolg eliminiert werden. Für Patienten mit persistierenden Inhibitoren bedeuten diese nicht nur eine drastische Verminderung der Lebensqualität sondern ein lebensbedrohliches Szenario. Eine Liganden-vermittelte Blockierung von neutralisierenden anti-FVIII Antikörpern sowie die zielgerichtete Ansteuerung des Rezeptors FVIII-spezifischer Gedächtnis-B-Zellen stellen mögliche Ansätze zur Verwirklichung antigenspezifischer ITI-Strategien für eine dauerhafte, vollständige Eliminierung von FVIII-Inhibitoren dar. Zu diesem Zweck wurden in dieser Arbeit durch Screening von phagenpräsentierten, randomisierten Peptidbibliotheken mit Inhibitor-positiven Patientenplasmen Peptidliganden selektioniert. Diese wiesen eine spezifische Bindung von anti-FVIII Antikörpern in den verwendten Plasmen auf. Durch den Einsatz entsprechender Software konnten AS-Konsensusmotive der Peptidsequenzen möglichen, konformationellen, funktionellen Inhibitorepitopen in der A2- sowie C2-Domäne von FVIII zugeordnet werden. Die von in silico-Analysen vorgegebene Domänenspezifität der anti-FVIII Antikörper wurde in Bindungsstudien mit rekombinant exprimierten FVIII-Domänen verfiziert. Die korrespondierenden, synthetischen Peptidliganden blockierten die IgG-Bindung an FVIII und regenerierten partiell dessen Aktivität im Plasma. Die Peptide stellten funktionelle Mimotope der möglichen Inhibitorepitope in der A2- und C2-Domäne dar. Da FVIII neutralisierende Antikörper zumeist Epitope in beiden Domänen erkennen, wurden die Mimotope kombiniert, was in einer noch effektiveren Blockierung von FVIII-Inhibitoren resultierte. Weiterhin wiesen Mimotopkombinationen Kreuzreaktivität mit anti-FVIII IgG in heterologen Patientenplasmen auf. Durch Fusion der Peptide an die Multimerisierungsdomäne der alpha-Kette des humanen C4-Bindeproteins konnten in Zellkultur heptamere Proteine generiert werden. Gegenüber den synthetischen Peptiden wiesen die Multimere aufgrund ihrer Multivalenz sowie der strukturellen Integrität eine deutlich verbesserte Blockierung von anti-FVIII IgG auf. Das Multimerisierungskonzept erlaubte ferner die Kombination unterschiedlicher Peptidliganden in einem Heteromultimer, was anhand der selektierten, funktionellen Mimotope für mögliche A2- und C2-Epitope getestet wurde. Weiterhin zeichneten sich die Inhibitor-spezifischen Multimere gegenüber den synthetischen Peptiden durch deutlich verlängerte Halbwerstzeiten aus. In Präparationen peripherer mononuklearer Zellen (PBMCs) von Patienten färbten synthetische Peptide sowie Fluoreszenz-markierter FVIII B-Zellsubpopulationen mit einem Gedächntis-B-Zell Phänotyp (CD19+IgG+). Gedächtnis-B-Zellen in PBMCs wurden polyklonal stimuliert. Im ELISPOT-Verfahren konnten Tetanusspezifische, jedoch keine FVIII-spezifischen Zellen, detektiert werden. Im Gegensatz zu den verwandten Kontrollen bewirkte eine Präinkubation der Zellen mit dem Peptid 12C6, welches an das toxische D-AS-Peptid (KLAKLAK)2 gekoppelt war, allerdings eine Reduktion von anti-FVIII IgG in den Überständen stimulierter Zellen

TIPT2 and geminin interact with basal transcription factors to synergize in transcriptional regulation

<p>Abstract</p> <p>Background</p> <p>The re-replication inhibitor Geminin binds to several transcription factors including homeodomain proteins, and to members of the polycomb and the SWI/SNF complexes.</p> <p>Results</p> <p>Here we describe the TATA-binding protein-like factor-interacting protein (TIPT) isoform 2, as a strong binding partner of Geminin. TIPT2 is widely expressed in mouse embryonic and adult tissues, residing both in cyto- and nucleoplasma, and enriched in the nucleolus. Like Geminin, also TIPT2 interacts with several polycomb factors, with the general transcription factor TBP (TATA box binding protein), and with the related protein TBPL1 (TRF2). TIPT2 synergizes with geminin and TBP in the activation of TATA box-containing promoters, and with TBPL1 and geminin in the activation of the TATA-less NF1 promoter. Geminin and TIPT2 were detected in the chromatin near TBP/TBPL1 binding sites.</p> <p>Conclusion</p> <p>Together, our study introduces a novel transcriptional regulator and its function in cooperation with chromatin associated factors and the basal transcription machinery.</p

Shift work and pathological conditions

Shift work exerts major influences on the physiological functions of the human body. These are primarily mediated by the disruption of circadian rhythms since most body functions are circadian rhythmic. Next to the disturbances caused by changes in the circadian system, shift work has also been suggested to be related to a number of other health disorders. The present study summarizes recently published data on the potential relationship between disorders and shift working

Non-Invasive Bleaching of the Human Lens by Femtosecond Laser Photolysis

Background: Globally, cataract is the leading cause of blindness and impaired vision. Cataract surgery is an attractive treatment option but it remains unavailable in sufficient quantity for the vast majority of the world population living in areas without access to specialized health care. Reducing blindness from cataract requires solutions that can be applied outside operating theatres. Cataract is a protein conformational disease characterized by accumulation of light absorbing, fluorescent and scattering protein aggregates. The aim of the study was to investigate whether these compounds were susceptible to photobleaching by a non-invasive procedure and whether this would lead to optical rejuvenation of the lens. Methodology/Principal Findings: Nine human donor lenses were treated with an 800 nm infra-red femtosecond pulsed laser in a treatment zone measuring 16160.52 mm. After laser treatment the age-induced yellow discoloration of the lens was markedly reduced and the transmission of light was increased corresponding to an optical rejuvenation of 3 to 7 years. Conclusions/Significance: The results demonstrate that the age-induced yellowing of the human lens can be bleached by a non-invasive procedure based on femtosecond laser photolysis. Cataract is a disease associated with old age. At the current technological stage, lens aging is delayed but with a treatment covering the entire lens volume complete optical rejuvenation is expected. Thus, femtosecond photolysis has the potential clinical value of replacing invasive cataract surgery by a non-invasive treatment modality that can be placed in mobile units, thus breaking down many of the barriers impedin

Challenges and opportunities for incentivising antibiotic research and development in Europe

Antimicrobial, and particularly antibiotic resistance are one of the world's biggest challenges today, and urgent action is needed to reinvigorate the antibiotic development pipeline. To inform policy discussions during and after the 2023 Swedish Presidency of the Council of the European Union, we critically appraise incentive options recently proposed by the European Commission, and member states, and consider what has been achieved over the last two decades in relation to antibiotic research and development. While several new antibiotics have achieved regulatory approval in recent years, almost none have innovative characteristics such as new chemical classes or novel mechanisms of action. We consider four incentive options to incentivise research and development of new antibiotics, including subscription payments, market entry rewards, transferable exclusivity extensions, and milestone payments. While each option has advantages and drawbacks, a combination of incentives may be required and continued investment is needed by the EU in push incentives, such as direct funding and grants, to incentivise drug discovery and preclinical stages of development. The EU must also coordinate with international initiatives and support access to new and pre-existing antibiotics in LMICs through platforms such as the WHO, and G7 and G20 group of countries

Assessment of left atrial functional parameters using a novel dedicated analysis tool for real-time three-dimensional echocardiography: validation in comparison to magnetic resonance imaging

Real-time three-dimensional echocardiography (RT3DE) is superior to two-dimensional echocardiography in assessing left atrial (LA) parameters, but to date algorithms developed for the left ventricle were applied due to a lack of dedicated LA software. In addition, no data are available on RT3DE assessment of active atrial contraction. The aim of this study was to validate a novel RT3DE analysis tool specifically dedicated to evaluate the LA. Cardiac magnetic resonance imaging (MRI) served as standard of reference. Fifty-five patients scheduled for pulmonary vein isolation underwent cardiac MRI and RT3DE. On ultrasound image datasets, a dynamic polyhedron model of the LA was generated from which LA maximum and minimum volumes (LAmax and LAmin), passive atrial emptying fraction (LAEF), and active atrial ejection fraction (LAEFtrue) were derived and compared to values obtained from cardiac MRI. High intraclass correlations between RT3DE and MRI were found for LAmax (r=0.94, p<0.001), LAmin (r=0.95, p<0.001), LAEF (r=0.92, p<0.001), and LAEFtrue (r=0.87, p<0.001). Similarly, Bland-Altman analysis revealed narrow limits of agreement for LAmax (−28.6 to 14.1ml), LAmin (−26.8 to 12.4ml), LAEF (−11.2 to 14.9%), and LAEFtrue (−10.6 to 6.8%). LAmax, LAmin and LAEFtrue were measured significantly (p<0.05) lower by RT3DE (111±38ml vs. 118±39ml, 73±38ml vs. 80±41ml, and 23±14% vs. 27±14%, respectively). Interobserver and intraobserver RT3DE measurements correlated closely. RT3DE using a novel dedicated software tool is valid, accurate and reproducible for assessing LA dimensional and functional parameters. This study corroborates previous reports and extends its validity to the assessment of active LA contractio

Soil nitrogen transformations under elevated atmospheric CO2 and O3 during the soybean growing season

We investigated the influence of elevated CO2 and O3 on soil N cycling within the soybean growing season and across soil environments (i.e., rhizosphere and bulk soil) at the Soybean Free Air Concentration Enrichment (SoyFACE) experiment in Illinois, USA. Elevated O3 decreased soil mineral N likely through a reduction in plant material input and increased denitrification, which was evidenced by the greater abundance of the denitrifier gene nosZ. Elevated CO2 did not alter the parameters evaluated and both elevated CO2 and O3 showed no interactive effects on nitrifier and denitrifier abundance, nor on total and mineral N concentrations. These results indicate that elevated CO2 may have limited effects on N transformations in soybean agroecosystems. However, elevated O3 can lead to a decrease in soil N availability in both bulk and rhizosphere soils, and this likely also affects ecosystem productivity by reducing the mineralization rates of plant-derived residues