| 1. |
- Engert, Andreas, et al.
(författare)
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The European Hematology Association Roadmap for European Hematology Research : a consensus document
- 2016
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Ingår i: Haematologica. - Pavia, Italy : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 101:2, s. 115-208
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Tidskriftsartikel (refereegranskat)abstract
- The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at (sic)23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine 'sections' in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients.
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| 2. |
- Alsafadi, Hani N, et al.
(författare)
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Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:12, s. 2718-2731
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Tidskriftsartikel (refereegranskat)abstract
- The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.
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| 3. |
- Augusto Silva, Iran, et al.
(författare)
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Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs
- 2023
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Ingår i: Frontiers in Biomaterials Science. - 2813-3749. ; 2-2023:1155919
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Tidskriftsartikel (refereegranskat)abstract
- Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable.
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| 4. |
- Boza-Serrano, Antonio, et al.
(författare)
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Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease
- 2019
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Ingår i: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 138:2, s. 251-273
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aβ) peptide, fibrillary tangles of intraneuronal tau and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer’s disease) mice and found specifically expressed in microglia associated with Aβ plaques. Single-nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aβ. Gal3 deletion decreased the Aβ burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aβ monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aβ aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2–DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.
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| 5. |
- De Santis, Martina M, et al.
(författare)
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Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue
- 2021
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Ingår i: Advanced Materials. - : Wiley. - 1521-4095 .- 0935-9648. ; 33:3
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.
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| 6. |
- Delaine, Tamara, et al.
(författare)
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Galectin-3-Binding Glycomimetics that Strongly Reduce Bleomycin-Induced Lung Fibrosis and Modulate Intracellular Glycan Recognition
- 2016
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227. ; 17:18, s. 1759-1770
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Tidskriftsartikel (refereegranskat)abstract
- Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone.
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| 7. |
- Ejserholm, Fredrik, et al.
(författare)
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Biocompatibility of a polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+) for neural implants.
- 2015
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Ingår i: Biomaterials research. - : Springer Science and Business Media LLC. - 2055-7124. ; 19:19, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- The flexibility of implantable neural probes has increased during the last 10 years, starting with stiff materials such as silicone to more flexible materials like polyimide. We have developed a novel polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+, consisting of a thiol, two allyls, an epoxy resin and two initiators), which is up to 100 times more flexible than polyimide. Since a flexible neural probe should be more biocompatible than a stiff probe, an OSTE+ probe should be more biocompatible than one composed of a more rigid material. We have investigated the toxicity of OSTE+ as well as of OSTE+ that had been incubated in water for a week (OSTE+H2O) using MTT assays with mouse L929 fibroblasts. We found that OSTE+ showed cytotoxicity, but OSTE+H2O did not. Extracts were analyzed using LC-MS and GC-MS in order to identify leaked chemicals.
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| 8. |
- Huang, Xiaoli, et al.
(författare)
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The Molecular Basis for Inhibition of Stemlike Cancer Cells by Salinomycin
- 2018
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Ingår i: ACS Central Science. - : American Chemical Society (ACS). - 2374-7943 .- 2374-7951. ; 4:6, s. 760-767
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Tidskriftsartikel (refereegranskat)abstract
- Tumors are phenotypically heterogeneous and include subpopulations of cancer cells with stemlike properties. The natural product salinomycin, a K+-selective ionophore, was recently found to exert selectivity against such cancer stem cells. This selective effect is thought to be due to inhibition of the Wnt signaling pathway, but the mechanistic basis remains unclear. Here, we develop a functionally competent fluorescent conjugate of salinomycin to investigate the molecular mechanism of this compound. By subcellular imaging, we demonstrate a rapid cellular uptake of the conjugate and accumulation in the endoplasmic reticulum (ER). This localization is connected to induction of Ca2+ release from the ER into the cytosol. Depletion of Ca2+ from the ER induces the unfolded protein response as shown by global mRNA analysis and Western blot analysis of proteins in the pathway. In particular, salinomycin-induced ER Ca2+ depletion up-regulates C/EBP homologous protein (CHOP), which inhibits Wnt signaling by down-regulating β-catenin. The increased cytosolic Ca2+ also activates protein kinase C, which has been shown to inhibit Wnt signaling. These results reveal that salinomycin acts in the ER membrane of breast cancer cells to cause enhanced Ca2+ release into the cytosol, presumably by mediating a counter-flux of K+ ions. The clarified mechanistic picture highlights the importance of ion fluxes in the ER as an entry to inducing phenotypic effects and should facilitate rational development of cancer treatments.
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| 9. |
- Langwiński, Wojciech, et al.
(författare)
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Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression
- 2022
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 147, s. 115-125
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation. Materials and methods: As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223–3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13. Results: We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223–3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223–3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes. Conclusions: These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223–3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223–3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.
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| 10. |
- Melo-Narváez, M. Camila, et al.
(författare)
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Lung regeneration : implications of the diseased niche and ageing
- 2020
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Ingår i: European Respiratory Review. - : European Respiratory Society (ERS). - 0905-9180 .- 1600-0617. ; 29:157
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Tidskriftsartikel (refereegranskat)abstract
- Most chronic and acute lung diseases have no cure, leaving lung transplantation as the only option. Recent work has improved our understanding of the endogenous regenerative capacity of the lung and has helped identification of different progenitor cell populations, as well as exploration into inducing endogenous regeneration through pharmaceutical or biological therapies. Additionally, alternative approaches that aim at replacing lung progenitor cells and their progeny through cell therapy, or whole lung tissue through bioengineering approaches, have gained increasing attention. Although impressive progress has been made, efforts at regenerating functional lung tissue are still ineffective. Chronic and acute lung diseases are most prevalent in the elderly and alterations in progenitor cells with ageing, along with an increased inflammatory milieu, present major roadblocks for regeneration. Multiple cellular mechanisms, such as cellular senescence and mitochondrial dysfunction, are aberrantly regulated in the aged and diseased lung, which impairs regeneration. Existing as well as new human in vitro models are being developed, improved and adapted in order to study potential mechanisms of lung regeneration in different contexts. This review summarises recent advances in understanding endogenous as well as exogenous regeneration and the development of in vitro models for studying regenerative mechanisms.
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| 11. |
- Nielsen, Morten Aagaard, et al.
(författare)
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Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- 4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL’s bioactivity.
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| 12. |
- Nielsen, Mathias Ingemann, et al.
(författare)
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Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:52, s. 20249-20262
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Tidskriftsartikel (refereegranskat)abstract
- Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.
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| 13. |
- Nowakowska, Joanna, et al.
(författare)
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Increased expression of ORMDL3 in allergic asthma : a case control and in vitro study
- 2023
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Ingår i: Journal of Asthma. - : Informa UK Limited. - 0277-0903 .- 1532-4303. ; 60:3, s. 458-467
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Asthma is the most frequent chronic disease in children. One of the most replicated genetic findings in childhood asthma is the ORMDL3 gene confirmed in several GWA studies in several paediatric populations.OBJECTIVES: The purpose of this study was to analyse ORMDL3 variants and expression in childhood asthma in the Polish population.MATERIALS AND METHODS: In the study we included 416 subject, 223 asthmatic children and 193 healthy control subjects. The analysis of two SNPs (rs3744246 and rs8076131) was performed using genotyping with TaqMan probes. The methylation of the ORMDL3 promoter was examined with Methylation Sensitive HRM (MS-HRM), covering 9 CpG sites. The expression of ORMDL3 was analysed in PBMCs from paediatric patients diagnosed with allergic asthma and primary human bronchial epithelial cells derived from healthy subjects treated with IL-13, IL-4, or co-treatment with both cytokines to model allergic airway inflammation.RESULTS: We found that ORMDL3 expression was increased in allergic asthma both in PBMCs from asthmatic patients as well as in human bronchial epithelial cells stimulated with the current cytokines. We did not observe significant differences between cases and controls either in the genotype distribution of analysed SNPs (rs3744246 and rs8076131) nor in the level of promoter methylation.CONCLUSIONS: Increased ORMDL3 expression is associated with paediatric allergic asthma and upregulated in the airways upon Th2-cytokines stimulation, but further functional studies are required to fully understand its role in this disease.
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| 14. |
- Ptasinski, Victoria, et al.
(författare)
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Targeting Alveolar Repair in Idiopathic Pulmonary Fibrosis
- 2021
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Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1535-4989. ; 65:4, s. 347-347
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Tidskriftsartikel (refereegranskat)abstract
- Idiopathic pulmonary fibrosis is a fatal interstitial lung disease with limited therapeutic options. Current evidence suggests that IPF may be initiated by repeated epithelial injury in the distal lung followed by abnormal wound healing responses which occur due to intrinsic and extrinsic factors. Mechanisms contributing to chronic damage of the alveolar epithelium in IPF include dysregulated cellular processes such as apoptosis, senescence, abnormal activation of developmental pathways, aging, as well as genetic mutations. Therefore, targeting the regenerative capacity of the lung epithelium is an attractive approach in the development of novel therapies for IPF. Endogenous lung regeneration is a complex process involving coordinated cross-talk between multiple cell types and re-establishment of a normal extracellular matrix environment. This review will describe the current knowledge of reparative epithelial progenitor cells in the alveolar region of the lung and discuss potential novel therapeutic approaches for IPF focusing on endogenous alveolar repair. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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| 15. |
- Stegmayr, John, et al.
(författare)
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Extracellular and intracellular small-molecule galectin-3 inhibitors
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.
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| 16. |
- Stegmayr, John
(författare)
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Galectin Inhibitors – Molecular Tools in Biomedical Research and Clinical Implications
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Galectins are β-galactoside-binding lectins, which are present in both the intracellular and extracellular environments of cells. During the last two decades, galectins have gained increasing interest as drug targets in various diseases, such as chronic inflammation (fibrosis) and cancer, in which galectins have been shown to play important, and sometimes rate-limiting, roles. Galectins have e.g. been implicated in cell-cell/matrix interactions, apoptosis, cell proliferation, cell migration, protein trafficking, and several immune modulatory functions. Because of this, development of galectin inhibitors has been of great importance, both as research tools to deepen our understanding of galectins in biological environments and as possible pharmaceuticals. In this thesis, two classes of galectin inhibitors are evaluated for these purposes, namely synthetic small-molecule galectin inhibitors and a heterogeneous group of plant-derived polysaccharides.The former inhibitor class has during the last decades been well-characterized for galectin inhibition in several biochemical in vitro assays, but are less investigated for biological activity (especially on a cellular level). To address this, we designed a novel assay for measurement of intracellular activity of galectin inhibitors, based on the phenomenon that galectins accumulate around disrupted vesicles. Furthermore, we show that it is possible to synthesize high-affinity galectin inhibitors with significant differences in cellular uptake, which thus may become valuable tools in the investigation of intracellular versus extracellular function of galectins. In another translational study of idiopathic pulmonary fibrosis, utilizing a bleomycin-induced lung fibrosis model in mice, we additionally show that one synthetic small-molecule galectin inhibitor may have clinical use in this disease.The plant-derived polysaccharide class of inhibitors has shown various biological activities and anti-cancer properties in cell cultures and in vivo. The mechanisms of these activities have, in a multitude of studies, been attributed to inhibition of galectins, mainly galectin-3, but proper biochemical analysis supporting this is missing. In this thesis, we show that this class of polysaccharides, in fact, is a very pore group of galectin inhibitors or in most cases does not inhibit the galectins at all.In conclusion, the work presented in this thesis will hopefully guide the development of future galectin inhibitors, to be used as molecular tools in galectin-related research and as potential therapeutics in the clinic.
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| 17. |
- Stegmayr, John, et al.
(författare)
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Isolation of high yield and quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts
- 2021
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Ingår i: American Journal of Physiology: Lung Cellular and Molecular Physiology. - : American Physiological Society. - 1522-1504 .- 1040-0605. ; 320:2, s. 232-240
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Tidskriftsartikel (refereegranskat)abstract
- Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared to animal models. Several experimental readouts have been established for use with PCLS, but obtaining high yield and quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for non-biased and high through-put analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis (IPF). We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.
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| 18. |
- Stegmayr, John, et al.
(författare)
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Low or No Inhibitory Potency of the Canonical Galectin Carbohydrate-binding Site by Pectins and Galactomannans
- 2016
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 291:25, s. 13318-13334
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Tidskriftsartikel (refereegranskat)abstract
- Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC50 values >10 mg/ml for a few or in most cases no inhibitory activity at all. The galactomannan Davanat® was more active, albeit not a strong inhibitor (IC50 values ranging from 3 to 20 mg/ml depending on the galectin). Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site.
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| 19. |
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| 20. |
- Wagner, Darcy E, et al.
(författare)
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Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Disease 2019
- 2020
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Ingår i: ERJ Open Research. - : European Respiratory Society (ERS). - 2312-0541. ; 6:4
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Forskningsöversikt (refereegranskat)abstract
- A workshop entitled "Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases" was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung. This selection of topics represents some of the most dynamic research areas in which incredible progress continues to be made. The workshop also included active discussion on the regulation and commercialisation of regenerative medicine products and concluded with an open discussion to set priorities and recommendations for future research directions in basic and translation lung biology.
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| 21. |
- Wasserstrom, Sebastian, et al.
(författare)
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Model visualization : from micro to macro
- 2022
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Ingår i: 3D lung models for regenerating lung tissue. - 9780323908719 ; , s. 207-221
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Bokkapitel (refereegranskat)abstract
- Because of increasing demand, rapid development of in vitro and in vivo models to be used to study lung regeneration and lung repair has occurred during the last years. Even if imaging has always been an important tool in diagnosing disease and validating models, the current disease models, including three-dimensional (3D) lung models, put a higher demand on advanced imaging techniques. Moreover, choosing the most relevant technique for a specific question is not a trivial task, and the rapid development of new techniques has not made this task easier. Therefore the aim of this chapter is to provide an overview of different advanced imaging techniques that can be used to evaluate and validate 3D lung models, to provide a discussion on the current state of the art, and to list the pros and cons of the available techniques.
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