A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation

The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.     

Sensory Neuron-Specific Deletion of Tropomyosin Receptor Kinase A (TrkA) in Mice Abolishes Osteoarthritis (OA) Pain via NGF/TrkA Intervention of Peripheral Sensitization

Tropomyosin receptor kinase A (TrkA/NTRK1) is a high-affinity receptor for nerve growth factor (NGF), a potent pain mediator. NGF/TrkA signaling elevates synovial sensory neuronal distributions in the joints and causes osteoarthritis (OA) pain. We investigated the mechanisms of pain transmission as to whether peripheral sensory neurons are linked to the cellular plasticity in the dorsal root ganglia (DRG) and are critical for OA hyperalgesia. Sensory neuron-specific deletion of TrkA was achieved by tamoxifen injection in 4-week-old TrkA^fl/fl;Na_V1.8^CreERT2 (Ntrk1 ^fl/fl;Scn10a^CreERT2) mice. OA was induced by partial medial meniscectomy (PMM) in 12-week-old mice, and OA-pain-related behavior was analyzed for 12 weeks followed by comprehensive histopathological examinations. OA-associated joint pain was markedly improved without cartilage protection in sensory-neuron-specific conditional TrkA knock-out (cKO) mice. Alleviated hyperalgesia was associated with suppression of the NGF/TrkA pathway and reduced angiogenesis in fibroblast-like synovial cells. Elevated pain transmitters in the DRG of OA-induced mice were significantly diminished in sensory-neuron-specific TrkA cKO and global TrkA cKO mice. Spinal glial activity and brain-derived neurotropic factor (BDNF) were significantly increased in OA-induced mice but were substantially eliminated by sensory-neuron-specific deletion. Our results suggest that augmentation of NGF/TrkA signaling in the joint synovium and the peripheral sensory neurons facilitate pro-nociception and centralized pain sensitization.     

Counteracting Immunosuppression in the Tumor Microenvironment by Oncolytic Newcastle Disease Virus and Cellular Immunotherapy

An apparent paradox exists between the evidence for spontaneous systemic T cell- mediated anti-tumor immune responses in cancer patients, observed particularly in their bone marrow, and local tumor growth in the periphery. This phenomenon, known as “concomitant immunity” suggests that the local tumor and its tumor microenvironment (TME) prevent systemic antitumor immunity to become effective. Oncolytic Newcastle disease virus (NDV), an agent with inherent anti-neoplastic and immune stimulatory properties, is capable of breaking therapy resistance and immunosuppression. This review updates latest information about immunosuppression by the TME and discusses mechanisms of how oncolytic viruses, in particular NDV, and cellular immunotherapy can counteract the immunosuppressive effect of the TME. With regard to cellular immunotherapy, the review presents pre-clinical studies of post-operative active-specific immunotherapy and of adoptive T cell-mediated therapy in immunocompetent mice. Memory T cell (MTC) transfer in tumor challenged T cell-deficient nu/nu mice demonstrates longevity and functionality of these cells. Graft-versus-leukemia (GvL) studies in mice demonstrate complete remission of late-stage disease including metastases and cachexia. T cell based immunotherapy studies with human cells in human tumor xenotransplanted NOD/SCID mice demonstrate superiority of bone marrow-derived as compared to blood-derived MTCs. Results from clinical studies presented include vaccination studies using two different types of NDV-modified cancer vaccine and a pilot adoptive T-cell mediated therapy study using re-activated bone marrow-derived cancer-reactive MTCs. As an example for what can be expected from clinical immunotherapy against tumors with an immunosuppressive TME, results from vaccination studies are presented from the aggressive brain tumor glioblastoma multiforme. The last decades of basic research in virology, oncology and immunology can be considered as a success story. Based on discoveries of these research areas, translational research and clinical studies have changed the way of treatment of cancer by introducing and including immunotherapy.     

The VEGF Inhibitor Soluble Fms-like Tyrosine Kinase 1 Does Not Promote AKI-to-CKD Transition

(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we studied whether transfection with sFlt1 aggravates experimental AKI-to-CKD transition and whether sFLT1 is increased in human kidney fibrosis. (2) Methods: Mice were transfected via electroporation with sFlt1. After confirming transfection efficacy, mice underwent unilateral ischemia/reperfusion injury (IRI) and were sacrificed 28 days later. Kidney histology and RNA were analyzed to study renal fibrosis, PTC damage and inflammation. Renal sFLT1 mRNA expression was measured in CKD biopsies and control kidney tissue. (3) Results: sFlt1 transfection did not aggravate renal fibrosis, PTC loss or macrophage recruitment in IRI mice. In contrast, higher transfection efficiency was correlated with reduced expression of pro-fibrotic and pro-inflammatory markers. In the human samples, sFLT1 mRNA levels were similar in CKD and control kidneys and were not correlated with interstitial fibrosis or PTC loss. (4) Conclusion: As we previously found that sFLT1 has therapeutic potential in diabetic nephropathy, our findings indicate that sFLT1 can be administered at a dose that is therapeutically effective in reducing inflammation, without promoting maladaptive kidney damage.     

Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin α_IIbβ₃ activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.     

Accuracy aspects in multiphase flow metering using X-raytransmission

The composition of a mixture of several components flowing through a pipe can be determined by means of an X-ray transmission technique/analysis. We present some aspects of the theory relating to an application in which the component volume fractions of an oil, water, and gas mixture are determined. It will be shown that on fundamental grounds, this technique has its limits regarding the number of components that can be distinguished in relation to the achieved accuracy of the measured attenuation     

Integral measurements with a plutonium inert matrix fuel rod in a heterogeneous light water reactor lattice

As part of PSI's validatory efforts for neutronics calculations pertaining to inert matrix fuel (IMF) deployment in light water reactors, first-of-their-kind integral measurements have been carried out at the PROTEUS facility employing a specially fabricated Pu-Er-Zr IMF rod in a heterogeneous (boiling water reactor) test lattice. Analogous experiments have also been conducted with MOX and “dummy” IMF rods, providing the basis for a systematic comparison of experimental and calculational results.     

Design and fabrication aspects of a plutonium-incineration experiment using inert matrices in a “Once-Through-Then-Out” mode

In the plutonium incineration experiment, named ‘Once-Through-Then-Out’ (OTTO), that is being prepared by JAERI, PSI and NRG, the use of highly stable inert matrices will be examined. The inert matrices MgAl₂O₄ spinel and ZrO₂ are insoluble in nitric acid and are considered as good storage media for final disposal. These inert matrices will be used in this experiment, which is representative for an OTTO scenario. A total of 7 Pu-containing targets were prepared for an irradiation in the High Flux Reactor in Petten. The objective of the irradiation is to reach a very high Pu-burnup. The main parameters to be studied are stability under irradiation, swelling, fission gas release and chemical interactions in the fuel. Four targets will be equipped with thermocouples for on-line monitoring of central temperature. Four of the targets contain MgAl₂O₄ as an inert matrix, 2 targets contain ZrO₂ and one target contains mixed-oxide (MOX) fuel for reference purposes. The fissile plutonium concentration is 0.32–0.44 g cm⁻³. Both particle-dispersed fuel and homogeneous dispersions were fabricated in order to test the effect of the size of the fissile inclusions. The design of the experiment and the fabrication of the samples are discussed.     

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