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Metastatic colorectal cancer (mCRC) remains one of the leading causes of cancer-related deaths worldwide with only a minority of patients responding to immune checkpoint targeting therapies. To effectively treat mCRC, we have developed a novel B7-H6/CD3 IgG-like T cell Engager (ITE) that mediates cytolytic synapse formation between T cells and tumor cells, re-directs their cytolytic activity selectively to B7-H6 (B7 homolog 6, NCR3LG1) expressing CRC cells, and increases infiltration of immune cells into the tumor tissue. B7-H6 is a member of the B7 family of immune receptors. We have identified B7-H6 as a novel antigen which is expressed in several solid tumor indications. In CRC tissues B7-H6 is expressed in >95 % of cases, while no to very little expression could be detected in normal tissues, thus making B7-H6 a promising antigen to re-direct cytolytic T cells to B7-H6-expressing CRC cells. The bispecific monovalent B7-H6/CD3 ITE is designed to bind simultaneously to CD3 on T cells and B7-H6 on CRC cells resulting in formation of a cytolytic synapse without interfering with the natural function of B7-H6 and has sustained serum exposure. In vitro, unstimulated T cells were co-cultured with B7-H6 expressing CRC cell lines and increasing concentrations of the B7-H6/CD3 ITE. The B7-H6/CD3 ITE induced dose-dependent lysis of CRC cell lines with EC50 values ranging from 0.9 to 25 ng/mL, whereas viability of B7-H6-negative cells was not affected, demonstrating the selectivity of the B7-H6/CD3 ITE for the B7-H6 antigen. In addition, the B7-H6/CD3 ITE induced B7-H6-dependent activation and proliferation of T cells, and granzyme B and cytokine secretion. In vivo, the B7-H6/CD3 ITE induced dose-dependent anti-tumor activity in subcutaneous CRC (NCI-H716, HCT-15, HT-29) xenograft tumor bearing immunodeficient mice which were reconstituted with human PBMCs or T cells. Tumor regressions were observed with single doses of < 0.5 mg/kg of B7-H6/CD3 ITE administered as i.v. injection, and anti-tumor activity could be further enhanced by q7d dosing. Consistent with the mode-of-action, the B7-H6/CD3 ITE led to a profound infiltration of both CD4+ and CD8+ T cell subsets into the tumor, which correlated with cleaved caspase 3 expression on tumor cells and tumor shrinkage. The inflammatory tumor microenvironment that was created by treatment with the B7-H6/CD3 ITE also led to an increase of PD-1 on T cells and PD-L1 on the tumor cells. The B7-H6/CD3 ITE shows cross-reactivity to both B7-H6 and CD3 of human and non-human primate origin respectively, and demonstrates antibody-like pharmacokinetic properties in a single dose study in cynomolgus monkeys. Our pre-clinical data demonstrate that the B7-H6/CD3 ITE is a highly B7-H6-selective and efficacious T cell engaging antibody with the ability to convert a non-inflamed into an inflamed tumor environment which supports clinical development. Citation Format: Susanne Hipp, Wei Zhang, Xiaoyun Liao, Aurelie Auguste, Annika Osswald, Kathrin Bauer, Christian Walterskirchen, Farzaneh H. Sayedian, Manuela Kellner, Craig Giragossian, Vladimir Voynov, Andrew E. Nixon, Paul J. Adam. A novel B7-H6/CD3 bispecific IgG-like T cell engaging antibody for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 56.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

The generation of reactive oxygen species (ROS) plays an important role for the maintenance of cellular processes and functions in the body. However, the excessive generation of oxygen radicals under pathological conditions such as acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) leads to increased endothelial permeability. Within this hallmark of ALI and ARDS, vascular microvessels lose their junctional integrity and show increased myosin contractions that promote the migration of polymorphonuclear leukocytes (PMNs) and the transition of solutes and fluids in the alveolar lumen. These processes all have a redox component, and this chapter focuses on the role played by ROS during the development of ALI/ARDS. We discuss the origins of ROS within the cell, cellular defense mechanisms against oxidative damage, the role of ROS in the development of endothelial permeability, and potential therapies targeted at oxidative stress.

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques.

Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.

© 2016 the American Physiological Society.Vascular cell hyperproliferation and metabolic reprogramming contribute to the pathophysiology of pulmonary arterial hypertension (PAH). An important cause of PAH in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To better characterize this disease course we studied early changes in pulmonary artery smooth muscle cell (PASMC) proliferation and metabolism using a unique ovine model of pulmonary overcirculation. Consistent with PAH in adults, PASMCs derived from 4-wk-old lambs exposed to increased PBF (shunt) exhibited increased rates of proliferation. While shunt PASMCs also exhibited significant decreases in mitochondrial oxygen consumption, membrane potential, and tricarboxylic acid (TCA) cycle function, suggesting a switch to Warburg metabolism as observed in advanced PAH in adults, they unexpectedly demonstrated decreased glycolytic lactate production, likely due to enhanced flux through the pentose phosphate pathway (PPP). This may be a response to the marked increase in NADPH oxidase (Nox) activity and decreased NADPH/NADP+ ratios observed in shunt PASMCs. Consistent with these findings, pharmacological inhibition of Nox activity preferentially slowed the growth of shunt PASMCs in vitro. Our results therefore indicate that PASMC hyperproliferation is observed early in the setting of pulmonary overcirculation and is accompanied by a unique metabolic profile that is independent of HIF-1α, PDHK1, or increased glycolytic flux. Our results also suggest that Nox inhibition may help prevent pulmonary overcirculation-induced PAH in children born with CHD.

Assessing alterations of the parenchymal architecture is essential in understanding fibrosing interstitial lung diseases. Here, we present a novel method to visualise fibrotic remodelling in human lungs and correlate morphological three-dimensional (3D) data with gene and protein expression in the very same sample. The key to our approach is a novel embedding resin that clears samples to full optical transparency and simultaneously allows 3D laser tomography and preparation of sections for histology, immunohistochemistry and RNA isolation. Correlating 3D laser tomography with molecular diagnostic techniques enables new insights into lung diseases. This approach has great potential to become an essential tool in pulmonary research.

In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice. DFG/Oc 23/9-3 DFG/Oc23/10-1 DFG/REBIRTH Swiss National Science Foundation (SNF) National Institutes of Health BMBF/DZL

Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth.The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12-30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded.Irrespective of the age of patients almost all isolated cells reached 40-60 generations with no correlation between maximal cell division potential and patient age. Cells from patients22 years showed a significantly faster doubling time than the cells from patients ≥22 years.The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences.

Imaging of biological samples necessitates high requirements on multi modal 3D imaging techniques. Lately, the range of application fields has extended from transparent biological samples up to biological compartments on intransparent objects. We introduce SLOT as an innovative and highly efficient tool for multi modal visualization by intrinsic and extrinsic contrast mechanisms in biological model organisms with sizes up to several millimeters. One aim is the exploration of SLOTs capability to image organs of biological model organisms. Therefore, intrinsic contrast mechanisms were addressed regarding their ability for visualizing and quantitating structural details within the murine lung. Additionally we present SLOT as a valuable tool for the in vitro structural and volumetric large scale investigation of biofilm formation on implants with sizes up to several millimeters.

New optical techniques have the potential to fill the gap between radiological and microscopic approaches to assess the lung's internal structure. Since its quantitative assessment requires unbiased sampling and measurement principles, imaging of the whole lung with sufficient resolution for visualizing details is important. To address this request, we applied scanning laser optical tomography (SLOT) for the three dimensional imaging of mouse lung ex vivo. SLOT is a highly efficient flourescence and transmission microscopy technique allowing for 3D imaging of specimen of sizes up to several millimeters. Previously fixed lung lobes and whole lungs were optically cleared and subsequently imaged with SLOT while making use of intrinsic contrast mechanisms like absorption and autofluorescence. Imaging of airways, blood vessels and parenchyma is demonstrated. Volumetric SLOT datasets of the lung's internal structure can be analyzed in any preferred planar orientation. Moreover, the sample preparation preserves microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, SLOT is a useful technique to visualize and survey the internal structure of mouse lung at different scales and with various contrast mechanisms. Potential applications of SLOT in lung research are e.g. quantitative phenotype analysis of mouse models of human lung disease in combination with stereological methods.

Biofilms – communities of microorganisms attached to surfaces – are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far. Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.