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Zusammenfassung Hintergrund Keimstrangstroma(SCST)- und Keimzelltumoren (MOGCT) sind seltene ovarielle Neoplasien, die häufig bei Jugendlichen oder prämenopausalen Patientinnen auftreten. Die Prognose ist in allen Stadien gut, weshalb psychisches Wohlbefinden, Lebensqualität, Fertilitätserhalt und Kinderwunsch zu relevanten Themen der Langzeitüberlebenden werden. Ziel der Arbeit Untersuchung der Prävalenz der fertilitätserhaltenden Therapie bei Patientinnen mit ovariellen Keimstrangstromatumoren und Keimzelltumoren und die Erfassung von Zusammenhängen mit Angst, Depression, Lebensqualität und Sexualität nach Therapie. Material und Methoden Die Current-Ovarian-geRM-cell-and-Sex-Cord-stromal-Tumour-Treatment-strategies(CORSETT)-Studie ist eine multizentrische, retro- und prospektiv beobachtende Kohortenstudie der Arbeitsgemeinschaft für Gynäkologische Onkologie (AGO) der Deutschen Krebsgesellschaft. Patientinnen nach Keimstrangstroma- oder Keimzelltumorbehandlung wurden gebeten, Fragebögen zu beantworten (Hospital Anxiety and Depression Scale [HADS], Sexual Activity Questionnaire [SAQ], European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire [EORTC QLQ-C30]). Im Fokus dieser Analyse standen der Stellenwert der fertilitätserhaltenden Operation sowie die Ausprägung und potenziellen Zusammenhänge mit Sexualität, Lebensqualität, Schmerz, Angst und Depressivität. Ergebnisse Von 355 eingeschlossenen Patientinnen hatten 152 Patientinnen die Fragebögen ausgefüllt und wurden in die Analysen eingeschlossen. 85 Patientinnen hiervon (56 %) wurden fertilitätserhaltend operiert. 34 % der Patientinnen hatten klinisch relevante Angst und Depressivität. Patientinnen nach fertilitätserhaltender Operation waren signifikant häufiger sexuell aktiv, hatten mehr sexuelles Verlangen und litten seltener unter Angst und Depressivität als Patientinnen ohne fertilitätserhaltende operative Therapie. Das Ausmaß von Angst und Depressivität korrelierte mit dem Schmerzniveau, unabhängig vom zeitlichen Abstand zur Krebsdiagnose. Diskussion Die fertilitätserhaltende operative Therapie sollte, wann immer onkologisch vertretbar, den Patientinnen mit SCST und MOGCT angeboten werden, um den Krebsüberlebenden bestmögliche Lebensqualität zu ermöglichen. Ein wiederholtes Screening auf Angst, Depressivität, Schmerzen und Probleme in der Sexualität sollte anhand von standardisierten Fragebögen bei allen Patientinnen durchgeführt werden.

Introduction The intention of this study was to evaluate the level of anxiety and depression of malignant ovarian germ cell (MOGCT) and sex cord stromal tumors (SCST) survivors and to identify possible alterable cofactors. Methods CORSETT was an observational, multicenter, mixed retrospective/prospective cohort study of the AGO Studygroup. Women who had been diagnosed with MOGCTs and SCSTs between 2001 and 2011 were asked to complete the Hospital Anxiety and Depression Scale (HADS) to evaluate distress. Predictors of distress (type of surgery, chemotherapy, time since diagnosis, recurrence, second tumor, pain) were investigated using multivariate linear regression analysis. Results 150 MOGCT and SCST patients with confirmed histological diagnosis completed the questionnaire median seven years after diagnosis. They had a HADS total score ≥ 13 indicating severe mental distress in 34% of cases. Patients after fertility-conserving surgery had lower probability of severe mental distress than those without fertility-conserving treatment (β = − 3.1, p = 0.04). Pain was associated with the level of distress in uni- and multivariate analysis (coef 0.1, p Discussion Severe mental distress was frequent in patients with MOGCT and SCST and the level of pain was associated with the level of distress. Fertility conserving therapy, however, was associated with less mental distress. Screening and treatment of pain and depression is required to improve mental well-being in survivors of MOGCT and SCST.

The intention of this study was to evaluate the level of anxiety and depression of malignant ovarian germ cell (MOGCT) and sex cord stromal tumors (SCST) survivors and to identify possible alterable cofactors.CORSETT was an observational, multicenter, mixed retrospective/prospective cohort study of the AGO Studygroup. Women who had been diagnosed with MOGCTs and SCSTs between 2001 and 2011 were asked to complete the Hospital Anxiety and Depression Scale (HADS) to evaluate distress. Predictors of distress (type of surgery, chemotherapy, time since diagnosis, recurrence, second tumor, pain) were investigated using multivariate linear regression analysis.150 MOGCT and SCST patients with confirmed histological diagnosis completed the questionnaire median seven years after diagnosis. They had a HADS total score ≥ 13 indicating severe mental distress in 34% of cases. Patients after fertility-conserving surgery had lower probability of severe mental distress than those without fertility-conserving treatment (β = - 3.1, p = 0.04). Pain was associated with the level of distress in uni- and multivariate analysis (coef 0.1, p 0.01, coef. Beta 0.5).Severe mental distress was frequent in patients with MOGCT and SCST and the level of pain was associated with the level of distress. Fertility conserving therapy, however, was associated with less mental distress. Screening and treatment of pain and depression is required to improve mental well-being in survivors of MOGCT and SCST.

AimsEnd-stage renal disease (ESRD) treated by chronic hemodialysis (HD) is associated with poor cardiovascular (CV) outcomes, with no available evidence-based therapeutics. A multiplexed proteomic approach may identify new pathophysiological pathways associated with CV outcomes, potentially actionable for precision medicine.Methods and resultsThe AURORA trial was an international, multicentre, randomized, double-blind trial involving 2776 patients undergoing maintenance HD. Rosuvastatin vs. placebo had no significant effect on the composite primary endpoint of death from CV causes, nonfatal myocardial infarction or nonfatal stroke. We first compared CV risk-matched cases and controls (n = 410) to identify novel biomarkers using a multiplex proximity extension immunoassay (276 proteomic biomarkers assessed with OlinkTM). We replicated our findings in 200 unmatched cases and 200 controls. External validation was conducted from a multicentre real-life Danish cohort [Aarhus-Aalborg (AA), n = 331 patients] in which 92 OlinkTM biomarkers were assessed. In AURORA, only N-terminal pro-brain natriuretic peptide (NT-proBNP, positive association) and stem cell factor (SCF) (negative association) were found consistently associated with the trial's primary outcome across exploration and replication phases, independently from the baseline characteristics. Stem cell factor displayed a lower added predictive ability compared with NT-ProBNP. In the AA cohort, in multivariable analyses, BNP was found significantly associated with major CV events, while higher SCF was associated with less frequent CV deaths.ConclusionsOur findings suggest that NT-proBNP and SCF may help identify ESRD patients with respectively high and low CV risk, beyond classical clinical predictors and also point at novel pathways for prevention and treatment.

The European existing building stock contributes to 40% of the total energy use and 36% of the CO2 emissions. To deal with the climate crisis, European climate and energy objectives were defined. By 2050, CO2 emissions should be cut to 80-95% compared to 1990 and all buildings must be energy-neutral. The North-Sea Region alone consists of 22 million outdated dwellings built between 1950 and 1985 that are in high need of renovation. Nowadays, the renovation industry applies mainly manual on-site renovation techniques, resulting in a low renovation pace, relatively high labour costs and a long duration. To tackle the urgent need for rapid renovations, six countries of the North-Sea Region collaborate to upscale the current renovation process in the Interreg project INDU-ZERO “Industrialization of house renovations toward energy-neutral”. The project focuses on modular prefabricated renovation packages with fully integrated HVAC technologies to arrive at energy-neutral dwellings. The project researches the possibilities of far-reaching automated and industrialized production processes. A smart factory blueprint will be designed to speed up the renovation pace to a target of 15,000 renovation packages per year per factory while cutting the current price with 50%. This contribution focusses on three main topics: material use, operational energy use and transport. Firstly, the reasoning behind the renovation package design is explained. Next, the packages are adopted on an archetype dwelling to document the thermal performance before and after renovation. Finally, the associated logistics are studied. To summarize each individual research in a blanket result, the environmental impact is determined and compared to the non-renovated dwelling.

Vor Abschluss ihrer fertilen Phase erkranken in Deutschland etwa 9000 Frauen pro Jahr an einer onkologischen Erkrankung. Die gewahlte Chemotherapie kann die spatere Fruchtbarkeit masgeblich beeintrachtigen. Die Entnahme von Ovarialgewebe zur Kyrokonservierung und spateren Retransplantation hat als Therapieoption zum Fertilitatserhalt mittlerweile einen festen Stellenwert. So kann bei Verlust der Fertilitat durch eine Chemotherapie diese ggf. wiederhergestellt werden. Die erste autologe Ovarialgewebstransplantation am Menschen wurde 2000 von Oktay et al. beschrieben. Im Jahr 2004 berichteten dann Donnez et al. weltweit erstmalig von einer Geburt nach Ovarialgewebstransplantation, und 2012 kam es in Deutschland zur Geburt eines gesunden Kindes nach Ovarialgewebstransplantation. In Deutschland werden mittlerweile jahrlich weit uber 300 Ovarialgewebsentnahmen im Rahmen des Netzwerks „FertiPROTEKT“ durchgefuhrt. Seit der Erfassung der Daten im FertiPROTEKT-Register wurden somit etwa 2500 Kryokonservierungen durchgefuhrt. Das Netzwerk „FertiPROTEKT“ von Gynakologen und Biologen aus dem deutschsprachigen Raum wurde 2006 gegrundet. Es hat sich zur Aufgabe gestellt, Frauen vor einer potenziell ovarschadigenden Therapie bezuglich fertilitatserhaltender Techniken zu beraten. Mittlerweile wird uber Schwangerschaftsraten von bis zu uber 33 % und eine Geburtenrate von 25 % nach Ovarialgewebsretransplantation berichtet. Naturlich mussen die individuellen Risiken individuell mit der Patientin besprochen werden. Somit hat die noch vor wenigen Jahren als experimentell zu betrachtende Kryokonservierung und Retransplantation von Ovarialgewebe einen festen Stellenwert in der Fertilitatsprotektion erhalten.

Hintergrund Die operative Therapie des Zervixkarzinoms ist stadienabhangig und sollte komplexen, aber exakt definierten Therapie-Algorithmen unter Berucksichtigung des Allgemeinzustands, des Tumortyps, der Tumorbiologie (und ggf. vorhandenen morphologischen Risikofaktoren) sowie der personlichen Lebens- und Familienplanung der Patientinnen folgen.

Background We have previously developed and confirmed a pragmatic molecular classifier for endometrial cancers; ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Inspired by the Cancer Genome Atlas, ProMisE identifies four prognostically distinct molecular subtypes and can be applied to diagnostic specimens (biopsy/curettings) enabling earlier informed decision-making. We have strictly adhered to the Institute of Medicine (IOM) guidelines for the development of genomic biomarkers, and herein present the final validation step of a locked-down classifier before clinical application. Patients and methods We assessed a retrospective cohort of women from the Tubingen University Women’s Hospital treated for endometrial carcinoma between 2003 and 2013. Primary outcomes of overall, disease-specific, and progression-free survival were evaluated for clinical, pathological, and molecular features. Results Complete clinical and molecular data were evaluable from 452 women. Patient age ranged from 29 to 93 (median 65) years, and 87.8% cases were endometrioid histotype. Grade distribution included 282 (62.4%) G1, 75 (16.6%) G2, and 95 (21.0%) G3 tumors. 276 (61.1%) patients had stage IA disease, with the remaining stage IB [89 (19.7%)], stage II [26 (5.8%)], and stage III/IV [61 (13.5%)]. ProMisE molecular classification yielded 127 (28.1%) MMR-D, 42 (9.3%) POLE, 55 (12.2%) p53abn, and 228 (50.4%) p53wt. ProMisE was a prognostic marker for progression-free (P = 0.001) and disease-specific (P = 0.03) survival even after adjusting for known risk factors. Concordance between diagnostic and surgical specimens was highly favorable; accuracy 0.91, κ 0.88. Discussion We have developed, confirmed, and now validated a pragmatic molecular classification tool (ProMisE) that provides consistent categorization of tumors and identifies four distinct prognostic molecular subtypes. ProMisE can be applied to diagnostic samples and thus could be used to inform surgical procedure(s) and/or need for adjuvant therapy. Based on the IOM guidelines this classifier is now ready for clinical evaluation through prospective clinical trials.

In order to ensure the quality of biomedical products, an experimental test for every single manufactured batch is required for many products. Especially in vaccine testing, animal experiments are traditionally used for this purpose. For example, efficacy is often determined via challenge experiments in laboratory animals. Safety tests of vaccine batches are also mostly performed using laboratory animals. However, many animal experiments have clear inherent disadvantages (low accuracy, questionable transferability to humans, unclear significance). Furthermore, for ethical reasons and animal welfare aspects animal experiments are also seen very critical by the public. Therefore, there is a strong trend towards replacing animal experiments with methods in which no animals are used ("replacement"). If a replacement is not possible, the required animal experiments should be improved in order to minimize the number of animals necessary ("reduction") and to reduce pain and suffering caused by the experiment to a minimum ("refinement"). This "3R concept" is meanwhile firmly established in legislature. In recent years many mandatory animal experiments have been replaced by alternative in vitro methods or improved according to the 3R principles; numerous alternative methods are currently under development. Nevertheless, the process from the development of a new method to its legal implementation takes a long time. Therefore, supplementary regulatory measures to facilitate validation and acceptance of new alternative methods could contribute to a faster and more consequent implementation of the 3R concept in the testing of biomedical products.

Background The presence of disseminated tumor cells (DTCs) in bone marrow of patients with early breast cancer (EBC) has been correlated with increased risk of metastatic disease or locoregional relapse. Zoledronic acid (ZOL) treatment has reduced DTCs in the bone marrow of patients with EBC in several studies. This controlled study sought to confirm these observations. Patients and methods Patients with EBC and DTC-positive bone marrow were randomized (N = 96) to treatment with ZOL plus adjuvant systemic therapy or adjuvant systemic therapy alone. The change in DTC numbers at 12 months versus baseline was measured. Results DTC-positive patients treated with ZOL were more likely to become DTC-negative after 12 months of treatment compared with the controls (67% versus 35%; P = 0.009). At 12 months, DTC counts decreased to a mean of 0.5 ± 0.8 DTCs in the ZOL group and to 0.9 ± 0.8 DTCs in the control group. In addition, ZOL was generally well tolerated. Conclusions Treatment with ZOL improves elimination of DTCs. Further studies are needed to determine whether the reduction in DTCs by ZOL provides clinical benefit.

Die wichtigsten Entwicklungen in der modernen operativen Gynakologie der letzten Jahre basieren auf der Minimierung des operativen Traumas durch elektrochirurgische Operationsverfahren und den minimal-invasiven Zugang. Die daraus resultierenden zunehmend komplexer werdenden minimal-invasiven Operationsverfahren erfordern eingebettete Operationssysteme zur Beherrschung der zentralen Schnittstelle nach intern und extern. Wahrend die Entwicklungen der letzten Jahre die funktionelle Ebene betrafen, so liegen die Schwerpunkte heute in der Verbesserung der Arbeitsablaufe und der Integration des Arbeitsplatzes Operationssaal in das Gesamtsystem Klinik. Masgebliche Parameter sind dabei die Erhohung der Patientensicherheit und die Verbesserung der Qualitatsstandards als Grundvoraussetzung fur Kosteneffizienz und Wirtschaftlichkeit zum Wohle der Patienten. Moderne minimal-invasive Operationsverfahren werden in Zukunft noch starker auf die Vorteile innovativer integrierter Operationssysteme (IOPS) angewiesen sein, wobei die weiteren Entwicklungsperspektiven noch ein groses Potenzial offenlassen, ganz besonders auch im Hinblick auf die Moglichkeiten der modernen Telemedizin.

Summary Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico- to nano-molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non-ideal confocal volume can be experimentally determined and validated.

The uptake dynamics of HCl by single sulfuric acid microdroplets under stratospheric conditions is investigated. The droplets are typically 30−70 μm in diameter and weakly charged so that they can be stored in an electrodynamic trap. The gas uptake of the droplets is monitored either by measuring their size by angle resolved Mie- scattering patterns or by electrostatic balancing of the droplets in the trap. At low temperatures and high sulfuric acid concentration (T < 190 K for 48 wt % H2SO4 and T < 195 K for 56 wt % H2SO4, respectively), liquid-phase diffusion inside the droplet is the rate-limiting step in the overall uptake process. In this regime, the diffusion coefficients Dliq of HCl in supercooled sulfuric acid solutions are found to increase strongly with temperature and H2O concentration. The results are discussed with respect to diffusion models that have been proposed recently. In contrast, at higher temperatures and lower sulfuric acid concentrations (30−40 wt % H2SO4, 185−207 K) gas-phase dif...

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ± 0.34 S/cm 2 ). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 × 10 6 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically “open” state with a mean single NPC electrical conductance of 1.7 ± 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

During the course of a human biomonitoring project (Biebesheim in Hessen, Germany) we elaborated a simple but sensitive method for the determination of tri- (TCP), tetra- (TeCP) and pentachlorophenol (PCP) in human urine. Urine samples, spiked with internal standards, were treated by acid hydrolysis. After a steam bath distillation the distillates were extracted using solid phase extraction. Derivatization of the chlorophenols was not carried out. GC/ECD system was used for detection. Detection limits of the chlorophenols were found in the range of 0.02 μg/L urine (detection limits of the ECD: 0.52 to 2.76 μg/L). By this method mono- and dichlorophenols cannot be detected. We investigated 24h-urine samples of 339 pupils (age 10 to 12 years). The children live either in the surroundings of a hazardous waste incinerator (SVA) in Biebesheim (n = 193), or controls (i.e. regions without waste incinerator) in the non polluted areas of Odenwald (n = 90) and Rheintal (n = 56). Between these three groups we did not find statistically significant differences in chlorophenol concentrations of the urine samples. The 95-percentiles of the analyzed samples are 0.74 μg/L (2,3,4-TCP), 1.24 μg/L (2,3,5-TCP), 0.70 μg/L (2,3,6–TCP), 1.10 μg/L (2,4,5–TCP), 1.74 μg/L (2,4,6–TCP), 2.84 μg/L (3,4,5–TCP), 4.78 μg/L (2,3,4,5-TeCP), 1.86 μg/L (2,3,4,6-TeCP), 2.90 μg/L (2,3,5,6-TeCP) and 4.39 μg/L (PCP).

Homogeneous nucleation rates are determined for micrometer sized water droplets levitated inside an electrodynamic Paul-trap. The size of a single droplet is continuously measured by analyzing the angle-resolved light scattering pattern of the droplets with classical Mie theory. The freezing process is detected by a pronounced increase in the depolarization of the scattered light. By statistical analysis of the freezing process of some thousand individual droplets, we obtained the homogeneous nucleation rate of water between 236 and 237 K. The values are in agreement with former expansion cloud chamber measurements but could be determined with considerably higher precision. The measurements are discussed in the light of classical nucleation theory in order to obtain the size and the formation energy of the critical nucleus.

A recombinant substitution mutant (recSerETI) of the Erythrina caffra trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable. Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Five repetitive cycles of guanidinium chloride (GdmCl) -induced denaturation–renaturation yield the native mutant protein with its inhibitory activity fully restored. The only difference between the wildtype and the mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein. The pH range of stability is shifted from pH 1–9.5, for wtETI, to pH 2–9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis. The mid-point of the thermal unfolding transition of the mutant protein is at ∼65°C, as compared to ∼75°C for the wildtype protein.

Homogeneous ice nucleation rates in single isolated micro droplets of water are determined under various atmospheric conditions inside an electrodynamic Paul-trap. The droplet size is measured by angle-re- solved detection of the scattered light from a Helium-Neon laser. The freezing process is detected by a sudden change of the depolarization ratio which is an indication for the formation of non spherical particles. The experimentally determined homogeneous ice nucleation rates JLS are JLS = (2.2 0.4)·10 8 cm -3 s -1 at T = (-37.2 0.15)°C and JLS = (5.6 1.0) ·10 7 cm -3 s -1 at T = (-37.7 0.15)°C. These values are much more precise as determined by measurements inside a cloud expansion chamber. The method of single particle obser- vation eliminates complications arising from these nucleation rate meas- urements. The results underline the theory that one single ice germ of certain size is sufficient to induce the freezing of the whole droplet. The depolarization ratio of the scattered intensity in the parallel and perpendicular polarization plane I s / Ip from the frozen particles exhibits a broad distribution with a mean value of I s / Ip= 0.3.

Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammation that has been implicated in the pathogenesis of devastating clinical syndromes including septic shock. We have investigated the role of a TNF-responsive phosphatidylcholine-specific phospholipase C (PC-PLC) for the cytotoxic and proinflammatory activity of TNF. We show here that the cytotoxicity signaled for by the so-called "death domain" of the p55 TNF receptor is associated with the activation of PC-PLC. The xanthogenate tricyclodecan-9-yl (D609), a specific and selective inhibitor of PC-PLC, blocked the cytotoxic action of TNF on L929 and Wehi164 cells. In vivo, D609 prevented both adhesion molecule expression in the pulmonary vasculature and the accompanying leukocyte infiltration in TNF-treated mice. More strikingly, D609 protects BALB/c mice from lethal shock induced either by TNF, lipopolysaccharide, or staphylococcal enterotoxin B. Together these findings imply PC-PLC as an important mediator of the pathogenic action of TNF, suggesting that PC-PLC may serve as a novel target for anti-inflammatory TNF antagonists.

Batch potency testing of rabies vaccines could be done by challenge, measurement of serum response or antigen quantification. Here, we show the development of a serological test that was successfully validated for use in batch release. The serological test is based on serum neutralization (SNT). The correlation to the NIH challenge was demonstrated by batches passing respectively failing equivalently in the NIH and SNT. The SNT provides information on immunogenicity and exhibits several advantages to the NIH: 1) SNT uses many fewer animals for batch release. 2) SNT allows quantitative information on the individual serum response, in contrast to the "dead"/"alive" interpretation of the NIH. 3) SNT is quicker than the NIH and needs fewer working hours. 4) SNT avoids the highly disturbing intra-cerebral injection and suffering from rabies for mice and spares the staff the emotional stress of massively harming animals.

European Pharmacopoeia (Ph. Eur.) monograph 0451 on Rabies vaccine (inactivated) for veterinary use describes an in vivo batch potency test that is based on the NIH test. This assay uses a large number of mice and results in a significant degree of suffering. In the interest of replacement, reduction and refinement of animal tests (3R) a serological potency assay for Rabies vaccine (inactivated) for animal use, developed and validated at the Paul-Ehrlich-Institut, has been assessed in a collaborative study organised by the EDQM (European Directorate for the Quality of MedicinesHealthCare). The goal was to demonstrate the wider transferability of the proposed assay and confirm its suitability. The study involved 13 laboratories and assessed 4 different vaccines from the EU market. Results of the study confirm that a limit test using a relatively small number of animals in a serological assay is possible, reproducible and reliable. The optimal number of animals per vaccine is product specific but may roughly be indicated to be between 8 and 10 for the products included in this study. Non-responders should be included in the analysis because they may reflect sub-potent vaccines. However, there may be a need to impose a maximum on the number of non-responders allowed for the reference vaccine as a monitor for assay validity. This assay provides a significant 3R improvement in terms of both the number of animals used and the amount of suffering entailed and provides a more reliable and reproducible assay format than the vaccination challenge assay. It also reduces the time required as compared to the vaccination challenge assay. It has been recommended to the Ph. Eur. group of experts 15V that this assay be included as an alternative to the batch potency assay in the Ph. Eur. monograph 0451.

The purpose of the study was to analyze sonographic criteria for the differentiation of benign and malignant breast lesions using real-time spatial compound imaging (CT) in combination with adaptive image processing (XRES).In a retrospective analysis of 460 patients, the sonographic criteria: shape, orientation, margin, echo pattern and posterior acoustic features were determined using CT and XRES. All investigations were performed using a 12 MHz linear transducer. The findings were classified according to the DEGUM criteria analogous to BIRADS and were histologically confirmed by core needle or vacuum biopsy. Statistical analysis was performed using a Chi-square test, logistic uni- and multivariate regression analysis and an ROC-curve analysis to detect the false-positive rate.All investigated diagnostic criteria were significant in the descriptive analysis (Chi-squared). The multivariate analysis showed that the criteria of irregular shape versus round, and not circumscribed margin versus circumscribed margin as well as the posterior acoustic features of enhanced versus reduced have a significant influence on the prediction of a malignant finding. The univariate analysis also showed a statistical significance using the indifferent and not parallel orientation versus parallel. To attain a detection rate of 95 %, a false-positive rate of 60 % must be expected.This study shows that when using CT in combination with XRES, the analyzed sonographic criteria for the differentiation of benign and malignant breast lesions are still of diagnostic value. In particular, the shape, margin and posterior sonographic features are important.

Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico- to nano-molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non-ideal confocal volume can be experimentally determined and validated.