Convolutional neural networks (CNNs) have been recognized as valuable tools for rapid quantitative analysis of morphological changes in toxicologic histopathology. We have assessed the performance of CNN-based (Halo-AI) mitotic figure detection in hepatocytes in comparison with detection by pathologists. In addition, we compared with Ki-67 and 5-bromodesoxyuridin (BrdU) immunohistochemistry labeling indices (LIs) obtained by image analysis. Tissues were from an exploratory toxicity study with a glycogen synthase kinase-3 (GSK-3) inhibitor. Our investigations revealed that (1) the CNN achieved similarly accurate but faster results than pathologists, (2) results of mitotic figure detection were comparable to Ki-67 and BrdU LIs, and (3) data from different methods were only moderately correlated. The latter is likely related to differences in the cell cycle component captured by each method. This highlights the importance of considering the differences of the available methods upon selection. Also, the pharmacology of our test item acting as a GSK-3 inhibitor potentially reduced the correlation. We conclude that hepatocyte cell proliferation assessment by CNNs can have several advantages when compared with the current gold standard: it relieves the pathologist of tedious routine tasks and contributes to standardization of results; the CNN algorithm can be shared and iteratively improved; it can be performed on routine histological slides; it does not require an additional animal experiment and in this way can contribute to animal welfare according to the 3R principles.
Abstract Introduction Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, represent a highly promising class of cancer therapeutics with a strong immune stimulatory potential. VSV-GP, a chimeric Vesicular Stomatitis Virus (VSV) pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) is a potent tumor cell-lysing agent and capable of jump-starting α-tumor immunity. The current study explores the immune promoting properties of VSV-GP and one of its cargo-armed, next generation variants with a T-cell/DC-directed MoA. It further investigates clinically relevant combination therapies with checkpoint inhibition (α-PD-1) as well as a “second mitochondria-derived activator of caspases“ mimetic (SMACm). Experimental Procedures Immune competent mice bearing established LLC1-IFNARKO, B16-F1-OVA or CT26.CL25-IFNARKO tumors were subject to treatments comprising VSV-GP variants, α-PD-1, a SMACm or combinations of the latter. Anti-tumor effects were determined by tumor growth inhibition and/or changes in overall survival. VSV-GP´s tumor cell selective replication, resulting in immune activation as well as cargo mediated immune modulation, were assessed using FFPE based IHC staining as well as NanoString and functional in vitro assays. Data Summary Here we present data on VSV-GP and one of its improved, next generation variants, which display potent α-tumor efficacy and upon systemic delivery increase the local T-cell and dendritic cell infiltration as well as activation within the tumor microenvironment. Building on the observed therapeutic and immune-modulatory potential of VSV-GP and its cargo-armed variant, we further explored therapeutic combinations with a PD-1 blocking antibody, which resulted in a strong improvement of the VSV-GP-mediated α-tumor efficacy. Immunological memory formation in cured animals was demonstrated by a tumor rechallange experiment. We further explored the therapeutic combination of VSV-GP and its cargo-bearing variant with a SMACm, which resulted in a strongly improved therapeutic benefit as well as increased overall survival in case of VSV-GP. The VSV-GP encoded immune-stimulatory cargo further improved this therapeutic synergy. Conclusion In summary VSV-GP induces a pro-inflammatory microenvironment within infected tumors, increases immune cell infiltration, a feature, which is further improved by tumor selective expression of VSV-GP encoded therapeutic cargos, and synergizes with α-PD-1 as well as a SMACm. The strong α-tumor immunity promoting potential of the VSV-GP platform observed in preclinical disease models justifies further testing of VSV-GP variants and the respective therapeutic combinations in clinical trials. Citation Format: Philipp Mueller, Tobias Nolden, Klaus Erb, Monika Petersson, Brigit Stierstorfer, Fabian Heinemann, Valerie Laura Herrmann, Maria Antonietta Impagnatiello, Eric Borges, Knut Elbers, John Park, Guido Wollmann, Patrik Erlmann. Therapeutic potential of cargo-armed next generation variants of VSV-GP and synergy with immune modulators [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4450.
Hintergrund: Multifokallinsen sind derzeit Methode der ersten Wahl für die Presbyopiekorrektur. Dabei ist die postoperative Emmetropie entscheidend für die Patientenzufriedenheit. Daher scheint ein zweizeitiges Vorgehen möglicherweise sinnvoll. Seit kurzem existiert eine diffraktive Add-On-Multifokallinse, die im Rahmen oder nach einer Kataraktoperation zusätzlich zu der Kapselsack-fixierten Monofokallinse eingesetzt werden kann. Unsere ersten Erfahrungen mit dieser Linse (6 Monate) stellen wir vor. Methode: Bei bisher 12 Patienten mit Wunsch nach Brillenunabhängigkeit wurde im Rahmen der Kataraktoperation bilateral zusätzlich eine diffraktive Add-On-Linse implantiert (3-stückig, Silikon, Nahzusatz +3,5dpt, Fa. 1stQ). Postoperativ untersuchten wir: Visus in verschiedenen Entfernungen, Defokussierkurve, Kontrastsehen und die subjektive Zufriedenheit. Ergebnisse: Die Ergebnisse entsprechen denen, die wir auch für eine Kapselsack-fixierte diffraktive Linse mit gleichem Nahzusatz erwarten. Der binokulare Fernvisus betrug im Median 1,0, der Nahvisus 0,85. Es zeigte sich eine hohe Patientenzufriedenheit. Eine Linse musste aufgrund einer postoperativen Dezentrierung explantiert werden.
Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6658.
Abstract Non-alcoholic fatty liver disease (NAFLD) and the progressive form of non-alcoholic steatohepatitis (NASH) are diseases of major importance with a high unmet medical need. Efficacy studies on novel compounds to treat NAFLD/NASH using disease models are frequently evaluated using established histological feature scores on ballooning, inflammation, steatosis and fibrosis. These features are assessed by a trained pathologist using microscopy and assigned discrete scores. We demonstrate how to automate these scores with convolutional neural networks (CNNs). Whole slide images of stained liver sections are analyzed using two different scales with four CNNs, each specialized for one of four histopathological features. A continuous value is obtained to quantify the extent of each feature, which can be used directly to provide a high resolution readout. In addition, the continuous values can be mapped to obtain the established discrete pathologist-like scores. The automated deep learning-based scores show good agreement with the trainer - a human pathologist.
Snap, crackle and pop! A simple method to obtain solvent-free, free-standing membranes suspended over apertures with 2.5 μm in diameter is described. The membranes are obtained by bursting of giant unilamellar vesicles (GUV) on a holey surface (see picture). Membrane integrity after bursting is fostered by enhancing the electrostatic interaction of the holey surface and the lipids.
Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).
As a first step towards transferring human grasping capabilities to robots, we analyzed the grasping behavior of human subjects. We derived a taxonomy in order to adequately represent the observed strategies. During the analysis of the recorded data, this classification scheme helped us to obtain a better understanding of human grasping behavior. We will provide support for our hypothesis that humans exploit compliant contact between the hand and the environment to compensate for uncertainty. We will also show a realization of the resulting grasping strategies on a real robot. It is our belief that the detailed analysis of human grasping behavior will ultimately lead to significant increases in robot manipulation and dexterity.
