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Crossing the blood–brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it.Significance:Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell–derived MMP9, which might enable the development of approaches to prevent brain metastasis.See related commentary by McCarty, p. 1167

Das FlexCAR besteht aus einer autonom gesteuerten Fahrzeugplattform für die Mobilität von morgen, welche Use-Case-gesteuert als Forschungsdemonstrator fungiert, um neue technologische Features unmittelbar aus dem Forschungsstadium nach dem Plug-and-Play–Prinzip gezielt zu implementieren. Damit kann eine frühzeitige Validierung im Hinblick auf ein künftiges Anwendungspotenzial ermöglicht werden. Offene Soft- und Hardwareschnittstellen sind hier berücksichtigt oder werden weiterentwickelt. FlexCAR comprises an autonomously controlled vehicle platform for the mobility of tomorrow. Use case-guided as a research platform, it enables implementing new technological features according to the plug-and-play principle. It allows for an early validation concerning potential future applications. Open software and hardware interfaces are included and will be further developed.

Crossing the blood–brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. Significance: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell–derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167

Objective This study was undertaken to calculate epilepsy‐related direct, indirect, and total costs in adult patients with active epilepsy (ongoing unprovoked seizures) in Germany and to analyze cost components and dynamics compared to previous studies from 2003, 2008, and 2013. This analysis was part of the Epi2020 study. Methods Direct and indirect costs related to epilepsy were calculated with a multicenter survey using an established and validated questionnaire with a bottom‐up design and human capital approach over a 3‐month period in late 2020. Epilepsy‐specific costs in the German health care sector from 2003, 2008, and 2013 were corrected for inflation to allow for a valid comparison. Results Data on the disease‐specific costs for 253 patients in 2020 were analyzed. The mean total costs were calculated at €5551 (±€5805, median = €2611, range = €274–€21 667) per 3 months, comprising mean direct costs of €1861 (±€1905, median = €1276, range = €327–€13 158) and mean indirect costs of €3690 (±€5298, median = €0, range = €0–€11 925). The main direct cost components were hospitalization (42.4%), antiseizure medication (42.2%), and outpatient care (6.2%). Productivity losses due to early retirement (53.6%), part‐time work or unemployment (30.8%), and seizure‐related off‐days (15.6%) were the main reasons for indirect costs. However, compared to 2013, there was no significant increase of direct costs (−10.0%), and indirect costs significantly increased (p

Objective To evaluate the incidence and risk factors of generalized convulsive seizure (GCS)-related fractures and injuries during video-EEG monitoring. Methods We analyzed all GCSs in patients undergoing video-EEG-monitoring between 2007 and 2019 at epilepsy centers in Frankfurt and Marburg in relation to injuries, falls and accidents associated with GCSs. Data were gathered using video material, EEG material, and a standardized reporting form. Results A total of 626 GCSs from 411 patients (mean age: 33.6 years; range 3–74 years; 45.0% female) were analyzed. Severe adverse events (SAEs) such as fractures, joint luxation, corneal erosion, and teeth loosening were observed in 13 patients resulting in a risk of 2.1% per GCS (95% CI 1.2–3.4%) and 3.2% per patient (95% CI 1.8–5.2%). Except for a nasal fracture due to a fall onto the face, no SAEs were caused by falls, and all occurred in patients lying in bed without evidence of external trauma. In seven patients, vertebral body compression fractures were confirmed by imaging. This resulted in a risk of 1.1% per GCS (95% CI 0.5–2.2%) and 1.7% per patient (95% CI 0.8–3.3%). These fractures occurred within the tonic phase of a GCS and were accompanied by a characteristic cracking noise. All affected patients reported back pain spontaneously, and an increase in pain on percussion of the affected spine section. Conclusions GCSs are associated with a substantial risk of fractures and shoulder dislocations that are not associated with falls. GCSs accompanied by audible cracking, and resulting in back pain, should prompt clinical and imaging evaluations.

This study was undertaken to quantify epilepsy-related costs of illness (COI) in Germany and identify cost-driving factors.COI were calculated among adults with epilepsy of different etiologies and severities. Multiple regression analysis was applied to determine any epilepsy-related and sociodemographic factors that serve as cost-driving factors.In total, 486 patients were included, with a mean age of 40.5 ± 15.5 years (range = 18-83 years, 58.2% women). Mean 3-month COI were estimated at €4911, €2782, and €2598 for focal, genetic generalized, and unclassified epilepsy, respectively. The mean COI for patients with drug-refractory epilepsy (DRE; €7850) were higher than those for patients with non-DRE (€4720), patients with occasional seizures (€3596), or patients with seizures in remission for1 year (€2409). Identified cost-driving factors for total COI included relevant disability (unstandardized regression coefficient b = €2218), poorer education (b = €2114), living alone (b = €2612), DRE (b = €1831), and frequent seizures (b = €2385). Younger age groups of 18-24 years (b = -€2945) and 25-34 years (b = -€1418) were found to have lower overall expenditures. A relevant disability (b = €441), DRE (b = €1253), frequent seizures (b = €735), and the need for specialized daycare (b = €749) were associated with higher direct COI, and poorer education (b = €1969), living alone (b = €2612), the presence of a relevant disability (b = €1809), DRE (b = €1831), and frequent seizures (b = €2385) were associated with higher indirect COI.This analysis provides up-to-date COI data for use in further health economics analyses, highlighting the high economic impacts associated with disease severity, disability, and disease-related loss of productivity among adult patients with epilepsy. The identified cost drivers could be used as therapeutic and socioeconomic targets for future cost-containment strategies.

Background Brain metastases (BM) are a frequent complication of malignant melanoma (MM), with limited treatment options and poor survival. Prevention of BM could be more effective and better tolerated than treating established BM in various conditions. Methods To investigate the temporospatial dynamics of PI3K/Akt/mTOR (PAM) pathway activation during BM formation and the preventive potential of its inhibition, in vivo molecular imaging with an Akt biosensor was performed, and long-term intravital multiphoton microscopy through a chronic cranial window in mice. Results In vivo molecular imaging revealed invariable PAM pathway activation during the earliest steps of brain colonization. In order to perform a long-term intravascular arrest and to extravasate, circulating MM cells needed to activate their PAM pathway during this process. However, the PAM pathway was quite heterogeneously activated in established human brain metastases, and its inhibition with the brain-penetrant PAM inhibitor GNE-317 resulted in only modest therapeutic effects in mice. In contrast, giving GNE-317 in preventive schedules that included very low doses effectively reduced the growth rate and number of BM in two MM mouse models over time, and led to an overall survival benefit. Longitudinal intravital multiphoton microscopy found that the first, rate-limiting steps of BM formation—permanent intravascular arrest, extravasation, and initial perivascular growth—are most vulnerable to dual PI3K/mTOR inhibition. Conclusion These findings establish a key role of PAM pathway activation for critical steps of early metastatic brain colonization and reveal its pharmacological inhibition as a potent avenue to prevent the formation of clinically relevant BM.

To further extent our understanding of aquatic vision, we introduce a complete optical model of a goldfish eye, which comprises all important optical parameters for the first time. Especially a spherical gradient index structure for the crystalline lens was included, thus allowing a detailed analysis of image quality, regarding spot size, and wavelength dependent aberration. The simulation results show, that our realistic eye model generates a sufficient image quality, with a spot radius of 4.9 mm which is below the inter cone distance of 5.5 mm. Furthermore, we optically simulate potential mechanical processes of accommodation and compare the results with contradictory findings of previous experimental studies. The quantitative simulation of the accommodation capacity shows that the depth of field is strongly dependent on the resting position and becomes significantly smaller when shorter resting positions are assumed. That means, to enable an extended depth perception with high acuity for the goldfish an adaptive, lens shifting mechanism would be required. In addition, our model allows a clear prediction of the expected axial lens-shift, which is necessary to ensure a sufficient resolution over a large object range.

The intensification of European agriculture leads to soil degradation, reduction of biodiversity and an increased economic risk for the farmers. An approach towards solving this problem is crop diversification and the optimized use of resources. Increasing agricultural efficiency/resilience through diversification and the associated falling environmental costs could contribute to the growth of the European agricultural sector by adapting the entire value chain.The EU-funded project DIVERFARMING (Horizon 2020 no 728003) aims to develop and deploy innovative farming and agribusiness models based on crop diversification. Germany is involved with a broad-based study in organic steep slope viticulture in Wawern (Saar Valley).A fundamental issue of steep slope viticulture is related to vegetation management below the vines. In order to overcome problems of soil erosion and soil organic matter depletion, an increasing number of winemakers is establishing cover crops such as grasses and legumes in driving lanes. On the contrary, the area underneath the vines is typically kept free of vegetation to avoid fungal diseases and competition on water. As cover crops do not benefit to the value chain and may compete with vines on water or have other adverse effects on vine performance, an alternative strategy for vegetation management underneath vines in steep slope viticulture is required.Therefore, intercropping vines with perennial herbs like Thyme and Oregano growing underneath is a promising cropping practice to address the abovementioned issues. Both herbs are economically valuable and originate from dry and warm environments, which are typical for most viticultural areas. Furthermore, their relatively low need for water and flat-growing habitus is assumed to be suitable to cover the soil underneath the vines in order to protect against erosion and suppress weeds without having adverse effects on vine growth and -health. They can be marketed directly or indirectly as a concentrate for cosmetics, perfumes, nutritional supplements and food.During the 5-year project, we will investigate impacts on and interactions between crops, soil ecological and physicochemical properties as well as erosion and emission of greenhouse gases to evaluate ecological benefits of crop diversification. Selected results obtained within the first two years of investigation will be presented.

Chloroplast manganese transporter 1 (CMT1) and photosynthesis-affected mutant 71 (PAM71) are two membrane proteins that function sequentially to mediate the passage of manganese across the chloroplast envelope and the thylakoid membrane. CMT1 and PAM71 belong to a small five-member protein family in Arabidopsis thaliana. The other three, photosynthesis-affected mutant 71 like 3 (PML3), PML4 and PML5 are not predicted to reside in chloroplast membranes. In this study, the subcellular localization of PML3:GFP, PML4:GFP and PML5:GFP was determined using transient and stable expression assays. PML3:GFP localizes to the Golgi apparatus, whereas PML4:GFP and PML5:GFP are found in the endoplasmic reticulum. We also examined patterns of PML3, PML4 and PML5 promoter activity. Although the precise expression pattern of each promoter was unique, all three genes were expressed in the leaf vasculature and in roots. Greenhouse grown single mutants pml3, pml4, pml5 and the pml4/pml5 double mutant did not exhibit growth defects, however an inspection of the root growth revealed a difference between pml3 and the other genotypes, including wild-type, in 500 &micro, M manganese growth conditions. Strikingly, overexpression of PML3 resulted in a stunted growth phenotype. Putative functions of PML3, PML4 and PML5 are discussed in light of what is known about PAM71 and CMT1.

A comprehensive schematic eye model of the hooded merganser is introduced for the first time to advance the understanding of amphibious vision. It is comprised of two different configurations, the first one modeling its visual system in air (unaccommodated state) and the second one representing the case where the eye is immersed in water (accommodated state). The model was designed using available data of former studies, image analysis and the implementation of feasible assumptions that serve as starting values. An optimization process incorporating an optical design program is used to vary the starting values with the aim of finding the setup offering the best acuity. The image quality was measured using the root-mean-square radius of the focal spot formed on the retina. The resulting schematic eye model comprises all relevant optical specifications, including aspherical geometrical parameters for cornea and lens, distances between the surfaces, the gradient index distribution of the lens, the retinal specifications and the object distance in both media. It achieves a spot radius of 4.20 μ m for the unaccommodated state, which meets the expectations derived by the mean ganglion cell density and comparison with other animals. In contrast, under water the determined spot radius of 11.48 µ m indicates an acuity loss. As well as enhancing our understanding of the vision of the hooded merganser, the schematic eye model may also serve as a simulation basis for examing similar animal eyes, such as the cormorant or other fish hunting birds.

Most protein-encoding genes in eukaryotes contain introns, which are interwoven with exons. Introns need to be removed from initial transcripts in order to generate the final messenger RNA (mRNA), which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides, which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5′ end and AG at the 3′ end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations, which have been known for years. Recently, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here, we expand systematic investigations of non-canonical splice site combinations in plants across eukaryotes by analyzing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences, such as an apparently increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts are a likely explanation for this observation, thus indicating annotation errors. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 small nuclear RNA (snRNA) isoform might allow the recognition of GA as a 5′ splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3′ splice site compared to the 5′ splice site across animals, fungi, and plants.

BackgroundIn addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organismArabidopsis thalianawere published during the last years. Also, a SMRT-based assembly of Landsbergerectahas been generated that identified translocation and inversion polymorphisms between two genotypes of the species.ResultsHere we provide a chromosome-arm level assembly of theA. thalianaaccession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate ade novoassembly. In addition, we assembled the chondrome and plastome sequences.ConclusionsDetailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements betweenA. thalianaaccessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. Thisde novoassembly extends the known proportion of theA. thalianapan-genome.

This paper gives a detailed overview of the first swing frequency and parameters that are impacting it. With increasing renewables connected to system via converters, inertia in the system as well as inertial response of the system has been decreasing. Therefore, the first swing frequency will have higher nadir/zenith in future. The parameters which describe typical first swing frequency and allow easier benchmarking of different cases are introduced. The paper deals further with sensitivity analysis where impact of different generation mix on the first swing frequency is analyzed i.e. purely hydro, gas or nuclear/coal as well as their mixture systems is investigated. Furthermore, the impact of governor control parameters, disturbance size and different penetration levels of renewables connected via power electronics will be analyzed. In the second part of paper, some of the mitigation solutions will be presented and analyzed. As one of innovative solutions, the SVC PLUS FS will be shortly described. At the end, impact on the first swing frequency of synchronous condensers and SVC PLUS FS on a real case of UK on 27.04.2014 will be presented, showing that SVC PLUS FS has significantly better impact on first swing frequency and is very grid supportive in fast frequency response.

A fovea, the retinal pitted invagination in the area centralis, is found in many vertebrates such as fish, reptiles, birds and in higher primates including humans. Because of the refractive index step between the retinal tissue and vitreous, the fovea defines an optically effective interface with aspherical characteristics, which influences the ray propagation to the receptor layer buried about 250 μm within the retinal layers [1]. In principle, two basic foveal shapes can be distinguished [2]. On the one hand, the convexiclivate type of fovea is a deep, funnel-shaped depression with a convex slope of the side walls and is observed in birds, some lizards, and certain deep-sea teleosts. On the other hand, the concaviclivate type of fovea is shallow and gradually shaped; the human fovea is of this type. Previous examinations on the influence of the fovea on the image-forming process have mainly been focused on the steep foveae types in which increasing magnification and directional-dependent distortion effects were predicted [3], [4], [5], [6], [7]. These investigations have been restricted to simple geometrical descriptions of the foveal shape, such as spheres and paraboloids. Real geometrical foveal structures from living creatures have not yet been examined. Moreover, the ray-trace considerations have been partly reduced to a few selected rays approaching the vitreo-retinal interface, and very limited numbers of incidence directions and wavelength-dependent effects have been investigated. Studies on the refracting characteristics of human foveae types are even more rare [2], [8]. C. Ross analyzed the optical functionality of the foveae of tarsiers, neglecting the influence of the anterior part of the eye and regarding only position shifts of three single rays. This contribution aims to answer the question on whether a comprehensive human eye model, which takes all optical components and their aberrations into account, leads to more detailed findings. In particular, field- and wavelength-dependent influences on spot sizes and positions, based on actual ray-bundle-cones are considered, which allow the estimation of aberration effects. With the current investigation, we address this issue by combining a realistic anatomical model of the anterior parts of the eye with real human fovea geometries. For the anterior eye, the human eye model of Liou and Brennan [9], with a gradient index profile of the crystalline lens, was used. To investigate the influence of actual fovea geometries on image formation, two selected and different foveal shapes (average and extraordinary), based on in vivo optical coherence tomography (OCT) measurements of a human eye were used. We analyzed the ray-optical behavior and implemented additional considerations that involve the variation of the observed field position, pupil diameter, chromatic effects, and especially the imaging behavior of peripheral colors in the receptor plane when the primary color is focused on this theoretical surface. These simulations validate and expand the knowledge about the correlation between aperture size and real and theoretical spot size, the effect of the foveal shape on either spot size and axial spot position, and, finally, the lateral spot position and color dependency of the incident angle.

OBJECTIVEThe treatment of patients with spinal stenosis and concurrent degenerative spondylolisthesis is controversial. Two large randomized controlled clinical trials reported contradictory results. The authors hypothesized that a substantial number of patients will show evidence of micro-instability after a sole decompression procedure.METHODSThis study was a retrospective analysis of all cases of lumbar spinal stenosis treated at the Frankfurt University Clinic (Universitätsklinik Frankfurt) from 2010 through 2013. Patients who had associated spondylolisthesis underwent upright MRI studies in flexion and extension for identification of subtle signs of micro-instability. Clinical outcome was assessed by means of SF-36 bodily pain (BP) and physical functioning (PF) scales.RESULTSA total of 21 patients were recruited to undergo upright MRI studies. The mean duration of follow-up was 65 months (SD 16 months). Of these 21 patients, 10 (47%) showed signs of micro-instability as defined by movement of > 4 mm on flexion/extension MRI. Comparison of mean SF-36 BP and PF scores in the group of patients who showed micro-instability versus those who did not showed no statistically significant difference on either scale.CONCLUSIONSThere seems to be a substantial subset of patients who develop morphological micro-instability after sole decompression procedures but do not experience any clinically significant effect of the instability.

A novel method for the automated detection of the outer choroid boundary within spectral-domain optical coherence tomography image data, based on an image model within the space of functions of bounded variation and the application of quadratic measure filters, is presented. The same method is used for the segmentation of retinal layer boundaries and proves to be suitable even for data generated without special imaging modes and moderate line averaging. Based on the segmentations, an automated determination of the central fovea region and choroidal thickness measurements for this and two adjacent 1-mm regions are provided. The quality of the method is assessed by comparison with manual delineations performed by five trained graders. The study is based on data from 50 children of the ages 8 to 13 that were obtained in the framework of the LIFE Child study at Leipzig University.

Implementing VSC HVDC links into a meshed interconnected AC system, as planned in Germany, requires adequate operational strategies that continuously provide the power set points to the DC links. In this paper an automated operational strategy is proposed for adapting the transmitted power of parallel DC links to the current system conditions, taking the line loadings and other parameters into account. The concepts consider two objectives for the operation: the optimization of the loading of AC lines and the minimization of the overall transmission losses. The automatic methods compute the power set point based on the voltage angle difference at the converter's connecting points. This paper describes the implementation of the control concepts and presents analysis of them for use of several quasi-parallel DC links. The case study of four selected scenarios with DC links embedded in a meshed AC grid shows good applicability when one, two or three DC links are operated simultaneously.

A significant expansion of the power grid in Germany can be reduced, if a certain amount of the annual renewable generation is curtailed. This paper presents an approach for an optimal dynamic curtailment of renewable energy sources in meshed distribution grids. Here, the particular power feed-in is limited only if the thermal constraint of a certain line is reached. The method is specifically directed to reduce the power, which causes the thermal constraint. Therefore, new distribution factors are formulated based on the AC power flow equation. The impact of the proposed method will be analyzed with a probabilistic approach on a real German high voltage distribution grid, while taking into account the further increase of renewable energy sources. The proposed method shows high potential for reducing and optimizing the classical grid extension. The cost of the German Energy Turnaround can be minimized since the curtailed energy is low compared to the reduced transport capacity.

In Germany, new HVDC links are planned for power transmission from the north to the south. One possibility is to schedule the operation of the HVDC links, which are to be embedded in the AC grid. Because the calculation via optimal power flow is time intensive and does not include the current grid situation, a simplified control method is introduced. It has the advantage that the HVDC power set point can be set automatically. Furthermore, another operation mode handles alert situations when an AC line loading is higher than intended and, therefore, adapts the power set point during (N-1)-conditions to avoid line overloadings. This paper proposes an automated method, which minimizes transmission losses while including the AC line loadings as limits for the HVDC power. A numerical case study shows the functionality of the proposed concepts and a detailed (N-1)-analysis investigates the behavior during unexpected line outages.

Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins. According to the concept, the bispecific antibody (scDbFAP×CD3) retargets T cells in a MHC-independent manner to tumor cells, providing an artificial first signal that allows the costimulatory antibody-ligand fusion proteins (B7.2-Db and scFv-4-1BBL) likewise targeted to the tumor cells to modulate the T-cell response. In our model system, the target cells coexpress the fibroblast activation protein (FAP) and endoglin as antigens. ScDbFAPCD3 and B7.2-Db are targeted to FAP although by different antibody moieties, whereas scFv-4-1BBL is directed against endoglin. ScDbFAPCD3-induced T-cell stimulation could be enhanced by the addition of either B7.2-Db or scFv-4-1BBL and even further by the combination of both as shown in terms of cytokine release (interleukin-2/interferon γ), proliferation and activation marker expression (CD25). By combined costimulation, overall T-cell population strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype. ScFv-4-1BBL-mediated costimulation of naive CD8+ T cells promoted the expansion and development of cytotoxic T cells with strong effector potential. Thus, combining a bispecific antibody with antibody-ligand fusion protein-mediated CD28 and 4-1BB costimulation in a targeted approach shows great potential to generate and shape an immune response at the tumor site. Therefore, the adaptation of this approach to other immune modulatory ligands and tumor-relevant targets seems to be promising.

In a single-blinded study, optical side-effects of a potential femtosecond (fs)-laser therapy in presbyopic human lenses were tested. Simulation of this therapy was carried out by applying fs-laser patterns into standard contact lenses (CL). In the first part of the study, the influence of the numerical aperture on optical side-effects was investigated by comparing a typical fs-LASIK configuration to a fs-presbyopia treatment (n = 11). The second part focused on a possible improvement of visual performance by comparing a regular grid pattern to a randomly chosen spacing of the laser spots (n = 16). Visual acuity was measured with ETDRS charts, contrast sensitivity with F.A.C.T. charts and mesopic vision with Mesotest II. Forward scattered light was measured with the C-Quant (both instruments: Oculus Optikgerate GmbH, Germany). A questionnaire detected subjective quality of vision. Differences between laser-treated and untreated CL and among the modifications were analyzed. The laser-treated and standard CL indicated no significant difference in visual acuity, contrast sensitivity and mesopic vision without glare. While wearing modified lenses with a regular grid, quality of vision decreased significantly by means of mesopic vision with glare and subjective straylight. These modifications also caused an impairment of subjective quality of vision. In contrast, there was no significant difference between the random pattern and standard CL. The increase of optical side-effects was reproducibly dependent on the geometry of the laser-structure. A randomized grid induced the least limitation. The study results are useful for planning possible laser-patterns in fs-laser therapy of the presbyopic lens.

We have investigated the staining patterns of primary and metastatic melanoma lesions using F8, L19 and F16. These three clinical-stage antibodies are currently being studied in clinical trials for the pharmacodelivery of cytokines or therapeutic radionuclides to neoplastic sites in patients with cancer. Frozen sections of 24 primary and 29 metastatic melanoma lesions were stained, using immunofluorescence procedures, with biotinylated preparations of the F8, L19 and F16 antibodies, which are specific to the alternatively spliced extra domain A and extra domain B domains of fibronectin and A1 domain of tenascin-C, respectively. Blood vessels were costained using von Willebrand factor-specific antibodies. In primary cutaneous melanoma lesions, F16 and F8 (but not L19) strongly stained the basal lamina at the interface between epidermis and dermis, with a strikingly complementary pattern. By contrast, metastatic melanoma lesions displayed a strong and diffuse pattern of immunoreactivity with all three antibodies. It was found that the extracellular matrix in melanoma undergoes extensive remodelling during the transition from primary to metastatic lesions. The intense staining of metastatic melanoma lesions by the F8, L19 and F16 antibodies provides a strong rationale for the use of these antibodies and their derivatives for the treatment of melanoma patients and possibly for the personalized choice of the best performing antibody in individual patients.

We generated and characterized novel antibody-cytokine fusion proteins ("immunocytokines") based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed "F8-mIL7") of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed "F8-mIL7-F8"), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio=16:1, 24h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.

DNA-encoded chemical libraries, i.e., collections of compounds individually coupled to distinctive DNA fragments serving as amplifiable identification barcodes, represent a new tool for the de novo discovery of small molecule ligands to target proteins of pharmaceutical interest. Here, we describe the design and synthesis of a novel DNA-encoded chemical library containing one million small molecules. The library was synthesized by combinatorial assembly of three sets of chemical building blocks using Diels-Alder cycloadditions and by the stepwise build-up of the DNA barcodes. Model selections were performed to test library performance and to develop a statistical method for the analysis of high-throughput sequencing data. A library selection against carbonic anhydrase IX revealed a new class of submicromolar bis(sulfonamide) inhibitors. One of these inhibitors was synthesized in the absence of the DNA-tag and showed accumulation in hypoxic tumor tissue sections in vitro and tumor targeting in vivo.

The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.

Acute myeloid leukemia (AML) is a rapidly progressing disease that is accompanied by a strong increase in microvessel density in the bone marrow. This observation prompted us to stain biopsies of AML and acute lymphoid leukemia (ALL) patients with the clinical stage human monoclonal antibodies F8 L19 and F16 directed against markers of tumor angiogenesis. The analysis revealed that the F8 and F16 antibodies strongly stained 70 of AML and 75 of ALL bone marrow specimens whereas chloroma biopsies were stained with all three antibodies. Therapy experiments performed in immunocompromised mice bearing human NB4 leukemia with the immunocytokine F8 IL2 [consisting of the F8 antibody fused to human interleukin 2 (IL 2)] mediated a strong inhibition of AML progression. This effect was potentiated by the addition of cytarabine promoting complete responses in 40 of treated animals. Experiments performed in immunocompetent mice bearing C1498 murine leukemia revealed long lasting complete tumor eradication in all treated mice. The therapeutic effect of F8 IL2 was mediated by both natural killer cells and CD8+ T cells whereas CD4 + T cells appeared to be dispensable as determined in immunodepletion experiments. The treatment of an AML patient with disseminated extramedullary AML manifestations with F16 IL2 (consisting of the F16 antibody fused to human IL 2 currently being tested in phase 2 clinical trials in patients with solid tumors) and low dose cytarabine showed significant reduction of AML lesions and underlines the translational potential of vascular tumor targeting antibody cytokine fusions for the treatment of patients with leukemia. Copyright © 2013 American Association for the Advancement of Science.

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mice.

The objective of this paper is a new accommodating opto-mechanical model of the aging human eye for basic simulations of presbyopia, especially of the lens. The lens, consisting of cortex and nucleus, of the aging human eye is mechanically simulated by a FEM model with the program ANSYS. The model results in physiologically correct parameters, and is used as input for a complete optical eye model, implemented in the software ZEMAX. The optical performance of the model corresponds fully with clinical data and the model represents the changes in the mechanical and optical parameters during accommodation and due to the aging process. It is suitable for optical and mechanical simulation; therefore, for example, different possible treatments for presbyopia can be simulated. Furthermore first investigations of stray light due to the laser treatment and their impact on visual performance are presented.

Conventional cytotoxic therapies of cancer often suffer from a lack of specificity, leading to a poor therapeutic index and toxicities to normal organs. The targeted delivery of bioactive molecules (cytokines, radionuclides, drugs, photosensitizer, etc.) by means of an antibody specific to tumor-associated markers of the tumor stroma and endothelium offers an elegant way to overcome these drawbacks. In fact, tumor markers of the endothelium and extracellular matrix are particularly attractive, because of their accessibility for intravenously administered agents, their genomic stability and the various therapeutic options they offer, ranging from direct tumor cell killing, to tumor infarction and to the recruitment of immune cells.

We investigated the therapeutic action of F8-IL2, a fusion protein consisting of the F8 antibody specific to the alternatively spliced extradomain-A of fibronectin, in diabody format and of human interleukin-2 in the Caki-1 (ATCC®) model of human renal cell carcinoma grafted subcutaneously in nude mice.F8-IL2 was cloned, expressed in CHO cells and purified to homogeneity. This immunocytokine was administered alone or combined with 3 standard drugs commonly used as therapy for kidney cancer, including sunitinib, sorafenib and interferon-α, in 2 sets of doses and treatment schedules.Neither F8-IL2 nor any other therapeutic agent cured tumor bearing mice when used as a single agent. The best therapeutic results were observed for the combination of sunitinib with F8-IL2 in a continuous administration schedule, which yielded a 28% cure rate and substantial tumor growth retardation.Considering that recombinant interleukin-2 based immunocytokines are now being investigated in several clinical trials in patients with cancer alone or combined with chemotherapy our preclinical results provide a motivation to study F8-IL2 combined with sunitinib in clinical trials in patients with kidney cancer.

European Journal of Nuclear Medicine and Molecular Imaging, 37 (8), ISSN:1619-7070, ISSN:1619-7089

Costimulation is an essential step in T-cell activation and hence, represents an important aspect in cancer immunotherapy. 4-1BB, a member of the tumor necrosis factor receptor family, has gained particular interest as a costimulatory molecule. Here, we investigated the potential of a targeted activation of 4-1BB-mediated costimulation at the tumor site by generating a recombinant antibody-cytokine fusion protein composed of a single-chain antibody fragment (scFv36) specific for the tumor stromal antigen fibroblast activation protein (FAP) and the extracellular domain of the 4-1BB ligand (4-1BBL). The scFv36-4-1BBL fusion protein is a homotrimeric molecule that binds specifically to FAP and the receptor 4-1BB. T-cell costimulation was demonstrated by interferon-gamma release of peripheral blood mononuclear cells cocultured with FAP-expressing HT1080 cells upon T-cell receptor triggering by monoclonal anti-CD3 antibody. Costimulatory activity of the scFv36-4-1BBL fusion protein was concentration dependent, ligand-specific, and substantially constrained to FAP-expressing target cell binding. Furthermore, scFv36-4-1BBL enhanced T-cell activation when the bispecific antibody scDb33CD3 (specific for FAP and CD3) was used as primary stimulus. Thus, target cell-dependent costimulation with scFv36-4-1BBL constitutes a new option to enhance T-cell activation by bispecific antibodies or antigen-dependent T-cell receptor triggering and should be useful to improve T cell-mediated antitumor responses.