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Background Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well. Methods In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R. Results FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3). When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4). Fig. 1 UMAP clustering of stimulated cells. Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma. Fig. 3 Relative population abundances with DA_edge R testing. Fig. 4 Relative state marker abundance within clusters with DS_limma testing. Conclusion Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.

Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p

Osteochondral explants harvested from different species are valuable preclinical ex vivo models for tissue engineering research. In this chapter, we describe the isolation of osteochondral plugs from bovine stifle joints, followed by defect creation, and plug preparation in a straightforward manner before mechanical loading using a compression and shear bioreactor. The method can be adapted to isolate osteochondral plugs from any animal species and to load explants in any type of bioreactor.

We describe a proteomics analysis to determine the molecular differences between normothermically perfused (normothermic machine perfusion, NMP) human kidneys with urine recirculation (URC) and urine replacement (UR). Proteins were extracted from 16 kidney biopsies with URC (n = 8 donors after brain death [DBD], n = 8 donors after circulatory death [DCD]) and three with UR (n = 2 DBD, n = 1 DCD), followed by quantitative analysis by mass spectrometry. Damage‐associated molecular patterns (DAMPs) were decreased in kidney tissue after 6 hours NMP with URC, suggesting reduced inflammation. Vasoconstriction was also attenuated in kidneys with URC as angiotensinogen levels were reduced. Strikingly, kidneys became metabolically active during NMP, which could be enhanced and prolonged by URC. For instance, mitochondrial succinate dehydrogenase enzyme levels as well as carbonic anhydrase were enhanced with URC, contributing to pH stabilization. Levels of cytosolic and the mitochondrial phosphoenolpyruvate carboxykinase were elevated after 24 hours of NMP, more prevalent in DCD than DBD tissue. Key enzymes involved in glucose metabolism were also increased after 12 and 24 hours of NMP with URC, including mitochondrial malate dehydrogenase and glutamic‐oxaloacetic transaminase, predominantly in DCD tissue. We conclude that NMP with URC permits prolonged preservation and revitalizes metabolism to possibly better cope with ischemia reperfusion injury in discarded kidneys., The authors perform a proteomics analysis to identify molecular differences between normothermically machine perfused and discarded human kidneys with urine recirculation and urine replacement using lactated Ringers.

Background The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), that activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019). Here, we aim to understand how IL-36RA mutations contribute to development and maintenance of intestinal inflammation in Crohn’s disease. Methods By whole-exome sequencing (WES) and subsequently targeted Sanger sequencing we identified a Crohn’s disease (CD) patient with a heterozygous missense mutation in IL-36RA (p.Ser113Leu). To understand how the mutation affects immune cells composition and function, we characterized peripheral blood mononuclear cells (PBMCs) of the patient by mass cytometry and measured cytokine levels in the serum by ELISA. In an available WES dataset of 177 CD patients, 47 ulcerative colitis (UC) patients and 34 healthy donors (HD), we searched for additional patients with IL-36RA mutations. Finally, we overexpressed the identified IL-36RA mutations in HEK cells and produced mutated recombinant proteins to study the effect of the mutations on protein expression and function. Results Our IL-36RA mutation-carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. In the patient, we detected increased levels of IL-36RA, IL-18 and IL-23 in the serum and an increased frequency of TH17 cells in the blood. These findings could hint towards an over-activation of the IL-36 signaling pathway in the IL-36RA mutated patient. In the available WES data, we identified two more CD patients with IL-36RA mutations (p.Leu133Ile, p.Pro76Leu), indicating that IL-36RA mutations are enriched in CD patients. By overexpressing the three identified mutations in HEK cells, we could demonstrate that two mutations (p.Ser113Leu and p.Pro76Leu) lead to a reduced protein expression. Also, our current data shows that the p.Ser113Leu mutant has a reduced capacity to antagonize IL-36 stimulation. However, further testing is required for the two other IL-36RA mutants. Conclusion Our data suggests that IL-36RA mutations are enriched in Crohn’s disease patients and should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease.

Recent literature on the role of patents in shaping competition between incumbents and new entrants shows mixed evidence, as patents can discourage entry into markets but may also encourage potential entrants by increasing profitability from research and development. The increasing use of patents as strategic weapons motivates this investigation of the impact of innovation on competition. In a case study of US pharmaceutical cardiovascular submarkets over the period 1988–1998, we use a panel probit model to study the impact of a firm’s patents and rivals’ patents in the firm’s decision to launch new products. Our results show that the number of a firm’s lagged patents encourages the firm’s entry with new products, while rivals’ initial stock of patents discourages entry, but more recent patents promote entry by opening new technological opportunities.

This article presents a comprehensive inventory of the urban vascular flora of the town of Palermo. The data were gathered from extensive field collections, from specimens kept in different herbaria, and publications of the last 30 years. The floristic catalogue includes all the vascular plants that occur spontaneously within the urban area. Thirteen taxa are endemic to Sicily and one of these, Clinopodium raimondoi, is exclusive to Palermo’s area. The inventory comprises 1052 taxa belonging to 119 families and 225 genera, and contains 170 taxa non-native to the Italian flora.

Two symmetrical diacylguanidines, triophamine (1) and limaciamine (2), have been found to occur in three Polyceridae nudibranchs from Canary Islands. These compounds were previously reported from taxonomically related species collected from distinct geographical areas. Due to the peculiar occurrence of 1 and 2 in Polyceridae nudibranchs and, in particular, exclusively in members of Polycerinae and Triophinae subfamilies, it should be suggested that these diacylguanidines are distinctive chemical markers of this group of nudibranchs.

Background The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), which activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019). However, it is not clear how IL-36 signaling contributes to the development of intestinal inflammation and which cell types are involved in this process. To shed more light on the role of IL-36 in intestinal inflammation, we took the opportunity to perform an in-depth characterization of a Crohn’s disease patient with a mutation in IL36RA and identify cell subsets responding to IL-36 stimulation. Methods By whole exome-sequencing of EDTA blood we identified in a Crohn’s disease patient a heterozygous mutation in IL36RA (p.Ser113Leu), which we confirmed by Sanger sequencing. By ELISA, we quantified IL-36RA serum levels in comparison to other Crohn’s disease (CD) patients and healthy donors (HD). Finally, we stimulated peripheral blood mononuclear cells (PBMCs) of the IL-36RA patient, CD patients, and HDs with ionomycin/PMA, LPS or IL-36α and performed deep immune profiling by mass cytometry with 35 markers. Results Our IL36RA mutation carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. As an individual treatment, autologous stem cell transplantation was performed, but did not lead to long-term improvement in symptoms. By ELISA, we found strongly elevated IL-36RA levels in the serum of our patient compared to CDs and HDs. Mass cytometry analysis revealed that the IL36RA mutation does not lead to major shifts in the immune cell composition of the blood, as the frequencies of B, T, NK, and myeloid cells in PBMCs of our patient were comparable to those in HDs and CDs. Furthermore, our data revealed that IL-36α stimulation mainly induced TNFα and IL-6 expression in myeloid cells as well as IFNγ expression in NK cells but did not affect T and B cells. Conclusion Our data is the first description of a Crohn’s disease patient with an IL36RA mutation and indicate that defects in IL-36 signaling should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease. Furthermore, we identified NK cells and myeloid cells as main responders to IL-36α stimulation. As next step, we plan to use recombinant produced, mutant IL-36RA in in-vitro assays with immune cells and fibroblasts to study the effect of the mutation on the IL-36RA function in more depth.

The paper presents an analytical inverse method for the identification of the distance and the entity of impulsive contaminations of rivers, based on the temporal moments of the concentration measured in a cross-section. The proposed method is benchmarked with reference to several small to large rivers and tested against the uncertainty of the governing parameters and the presence of noise in the measures. In most of the considered applications, the method provides reliable solutions at a very limited computational cost.