The CD40 agonist HERA-CD40L results in enhanced activation of antigen presenting cells, promoting an anti-tumor effect alone and in combination with radiotherapy.

Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.

Active Remodeling of Capillary Endothelium via Cancer Cell-Derived MMP9 Promotes Metastatic Brain Colonization.

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.

Trends in resource utilization and cost of illness in patients with active epilepsy in Germany from 2003 to 2020.

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 < .028, +35.1%), resulting in a significant increase in total epilepsy-related costs (p < .047, +20.2%). Compared to the 2013 study population, a significant increase of cost of illness could be observed (p = .047). SIGNIFICANCE: The present study shows that disease-related costs in adult patients with active epilepsy increased from 2013 to 2020. As direct costs have remained constant, this increase is attributable to an increase in indirect costs. These findings highlight the impact of productivity loss caused by early retirement, unemployment, working time reduction, and seizure-related days off.

Multicenter, cross-sectional study of the costs of illness and cost-driving factors in adult patients with epilepsy.

OBJECTIVE: This study was undertaken to quantify epilepsy-related costs of illness (COI) in Germany and identify cost-driving factors. METHODS: 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. RESULTS: 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 for >1 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. SIGNIFICANCE: 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.

The PI3K/Akt/mTOR pathway as a preventive target in melanoma brain metastasis.

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.

Nintedanib and a bi-specific anti-VEGF/Ang2 nanobody selectively prevent brain metastases of lung adenocarcinoma cells.

Brain metastases (BM) are an ever-increasing challenge in oncology, threatening quality of life and survival of many cancer patients. The majority of BM originate from lung adenocarcinoma, and stage III patients have a risk of 40-50% to develop BM in the first years of disease onset. As therapeutic options are limited, prevention of their occurrence is an attractive concept. Here we investigated whether Nintedanib (BIBF 1120), a tyrosine kinase inhibitor (TKI) targeting the VEGF pathway approved for lung adenocarcinoma, and the dual anti-VEGF-A/Ang2 nanobody BI836880 have the potential to prevent BM formation. A mouse model of brain metastasis from lung adenocarcinoma was used in which tumor cells were injected intracardially. Metastases formation occurred inside and outside of the brain and was followed by MRI, IVIS, and immunohistochemistry. BM were reduced in volume and number by both Nintedanib and the dual anti-VEGF-A/Ang2 nanobody, which translated into improved survival. Both compounds were able to normalize cerebral blood vessels at the site of brain metastatic lesions. Extracranial metastases, however, were not reduced, and meningeal metastases only partially. Interestingly, unspecific control IgG also lead to brain vessel normalization and reduction of brain and meningeal metastases. This data indicates a brain-specific group effect of antiangiogenic compounds with respect to metastasis prevention, most likely by preventing an early angiogenic switch. Thus, Nintedanib and BI836880 are promising candidates for future BM preventive study concepts in lung adenocarcinoma patients.

Amphibious vision - Optical design model of the hooded merganser eye.

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 µm 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 µm 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.

Risk incidence of fractures and injuries: a multicenter video-EEG study of 626 generalized convulsive seizures.

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.

Homologous Proteins of the Manganese Transporter PAM71 Are Localized in the Golgi Apparatus and Endoplasmic Reticulum.

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 µM 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.

Animal, Fungi, and Plant Genome Sequences Harbor Different Non-Canonical Splice Sites.

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 Eurytemoraaffinis and Oikopleuradioica. 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.

HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of FcγR-binding functionality.

BACKGROUND: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. METHODS: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. RESULTS: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. CONCLUSION: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.

Upright MRI after decompression of spinal stenosis and concurrent spondylolisthesis.

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 chromosome-level sequence assembly reveals the structure of the Arabidopsis thaliana Nd-1 genome and its gene set.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession 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 a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions 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. This de novo assembly extends the known proportion of the A. thaliana pan-genome.

Comprehensive optical design model of the goldfish eye and quantitative simulation of the consequences on the accommodation mechanism.

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 µm which is below the inter cone distance of 5.5 µm. 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.

Automated detection of the choroid boundary within OCT image data using quadratic measure filters.

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.

Antibody-based delivery of interleukin-2 to neovasculature has potent activity against acute myeloid leukemia.

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.

Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

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.

Analysis of optical side-effects of fs-laser therapy in human presbyopic lens simulated with modified contact lenses.

BACKGROUND: 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). METHODS: 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 Optikgeräte GmbH, Germany). A questionnaire detected subjective quality of vision. Differences between laser-treated and untreated CL and among the modifications were analyzed. RESULTS: 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. CONCLUSION: 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.

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate.

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.

Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

BACKGROUND: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. CONCLUSION/SIGNIFICANCE: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.