| 1. |
- Alaridah, Nader, et al.
(författare)
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Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs
- 2017
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 9, s. 318-329
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.
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| 2. |
- Ambite, Ines, et al.
(författare)
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Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets
- 2016
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 12:10
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Tidskriftsartikel (refereegranskat)abstract
- Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. Trial Registration: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov).
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| 3. |
- Ambite, Ines, et al.
(författare)
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Susceptibility to urinary tract infection : Benefits and hazards of the antibacterial host response
- 2016
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Ingår i: Microbiology spectrum. - Washington, DC, USA : ASM Press. - 2165-0497. ; 4:3
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Forskningsöversikt (refereegranskat)abstract
- A paradigm shift is needed to improve and personalize the diagnosis of infectious disease and to select appropriate therapies. For many years, only the most severe and complicated bacterial infections received more detailed diagnostic and therapeutic attention as the efficiency of antibiotic therapy has guaranteed efficient treatment of patients suffering from the most common infections. Indeed, treatability almost became a rationale not to analyze bacterial and host parameters in these larger patient groups. Due to the rapid spread of antibiotic resistance, common infections like respiratory tract- or urinary-tract infections (UTIs) now pose new and significant therapeutic challenges. It is fortunate and timely that infectious disease research can offer such a wealth of new molecular information that is ready to use for the identification of susceptible patients and design of new suitable therapies. Paradoxically, the threat of antibiotic resistance may become a window of opportunity, by encouraging the implementation of new diagnostic and therapeutic approaches. The frequency of antibiotic resistance is rising rapidly in uropathogenic organisms and the molecular and genetic understanding of UTI susceptibility is quite advanced. More bold translation of the new molecular diagnostic and therapeutic tools would not just be possible but of great potential benefit in this patient group. This chapter reviews the molecular basis for susceptibility to UTI, including recent advances in genetics, and discusses the consequences for diagnosis and therapy. By dissecting the increasingly well-defined molecular interactions between bacteria and host and the molecular features of excessive bacterial virulence or host-response malfunction, it is becoming possible to isolate the defensive from the damaging aspects of the host response. Distinguishing "good" from "bad" inflammation has been a long-term quest of biomedical science and in UTI, patients need the "good" aspects of the inflammatory response to resist infection while avoiding the "bad" aspects, causing chronicity and tissue damage.
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| 4. |
- Andersson, Madelene, et al.
(författare)
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Development of an experimental ex vivo wound model to evaluate antimicrobial efficacy of topical formulations
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:9
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Tidskriftsartikel (refereegranskat)abstract
- Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and repro-ducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, P. aeruginosa and S. aureus. We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains success-fully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of an-tibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indi-cating that the demonstrated method mirrors typical in vivo infections.
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| 5. |
- Butler, Daniel S.C., et al.
(författare)
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Neuroepithelial control of mucosal inflammation in acute cystitis
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- The nervous system is engaged by infection, indirectly through inflammatory cascades or directly, by bacterial attack on nerve cells. Here we identify a neuro-epithelial activation loop that participates in the control of mucosal inflammation and pain in acute cystitis. We show that infection activates Neurokinin-1 receptor (NK1R) and Substance P (SP) expression in nerve cells and bladder epithelial cells in vitro and in vivo in the urinary bladder mucosa. Specific innate immune response genes regulated this mucosal response, and single gene deletions resulted either in protection (Tlr4−/− and Il1b−/− mice) or in accentuated bladder pathology (Asc−/− and Nlrp3−/− mice), compared to controls. NK1R/SP expression was lower in Tlr4−/− and Il1b−/− mice than in C56BL/6WT controls but in Asc−/− and Nlrp3−/− mice, NK1R over-activation accompanied the exaggerated disease phenotype, due, in part to transcriptional de-repression of Tacr1. Pharmacologic NK1R inhibitors attenuated acute cystitis in susceptible mice, supporting a role in disease pathogenesis. Clinical relevance was suggested by elevated urine SP levels in patients with acute cystitis, compared to patients with asymptomatic bacteriuria identifying NK1R/SP as potential therapeutic targets. We propose that NK1R and SP influence the severity of acute cystitis through a neuro-epithelial activation loop that controls pain and mucosal inflammation.
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| 6. |
- Chaturvedi, Swasti, et al.
(författare)
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Slit2 Prevents Neutrophil Recruitment and Renal Ischemia-Reperfusion Injury : english
- 2013
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Ingår i: Journal of the American Society of Nephrology. - 1046-6673. ; 24:8, s. 1274-1287
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.
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| 7. |
- Dahlman, Anna, et al.
(författare)
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Thrombin-derived C-terminal peptide reduces Candida-induced inflammation and infection in vitro and in vivo
- 2021
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804. ; 65:11
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Tidskriftsartikel (refereegranskat)abstract
- Infections due to the opportunistic fungus Candida have been on the rise in the last decades, especially in immunocompromised individuals and hospital settings. Unfortunately, the treatments available today are limited. Thrombin-derived C-terminal peptide (TCP-25) is an antimicrobial peptide (AMP) with antibacterial and immunomodulatory effects. In this work, we, for the first time, demonstrate the ability of TCP-25 ability to counteract Candida in vitro and in vivo. Using a combination of viable count assay (VCA), radial diffusion assay (RDA), and fluorescence and transmission electron microscopy analyses, TCP-25 was found to exert a direct fungicidal activity. An inhibitory activity of TCP-25 on NF-kB activation induced by both zymosan alone and heat-killed C. albicans was demonstrated in vitro using THP-1 cells, and in vivo using NF-kB reporter mice. Moreover, the immunomodulatory property of TCP-25 was further substantiated in vitro by analyzing cytokine responses in human blood stimulated with zymosan, and in vivo employing a zymosan-induced peritonitis model in C57BL/6 mice. The therapeutic potential of TCP-25 was demonstrated in mice infected with luminescent C. albicans. Finally, the binding between TCP-25 and zymosan was investigated using circular dichroism spectroscopy and intrinsic fluorescence analysis. Taken together, our results show that TCP-25 has a dual function by inhibiting Candida as well as the associated zymosan-induced inflammation. The latter function is accompanied by a change in secondary structure upon binding to zymosan. TCP-25, therefore, shows promise as a novel drug candidate against Candida infections.
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| 8. |
- Godaly, Gabriela, et al.
(författare)
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Urinary Tract Infection Molecular Mechanisms and Clinical Translation.
- 2016
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Ingår i: Pathogens. - : MDPI AG. - 2076-0817. ; 5:1
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Forskningsöversikt (refereegranskat)abstract
- Rapid developments in infection biology create new and exciting options for individualized diagnostics and therapy. Such new practices are needed to improve patient survival and reduce morbidity. Molecular determinants of host resistance to infection are being characterized, making it possible to identify susceptible individuals and to predict their risk for future morbidity. Immunotherapy is emerging as a new strategy to treat infections worldwide and controlled boosting of the host immune defense represents an important therapeutic alternative to antibiotics. In proof of concept studies, we have demonstrated that this approach is feasible. The long-term goal is not just to remove the pathogens but to also develop technologies that restore resistance to infection in disease-prone patients and devise personalized therapeutic interventions. Here, we discuss some approaches to reaching these goals, in patients with urinary tract infection (UTI). We describe critical host signaling pathways that define symptoms and pathology and the genetic control of innate immune responses that balance protection against tissue damage. For some of these genes, human relevance has been documented in clinical studies, identifying them as potential targets for immune-modulatory therapies, as a complement to antibiotics.
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| 9. |
- Ho, Chin Shing, et al.
(författare)
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Targeting of nucleotide-binding proteins by HAMLET-a conserved tumor cell death mechanism.
- 2015
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 1476-5594 .- 0950-9232.
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Tidskriftsartikel (refereegranskat)abstract
- HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.Oncogene advance online publication, 1 June 2015; doi:10.1038/onc.2015.144.
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| 10. |
- Ho Cs, James, et al.
(författare)
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HAMLET: functional properties and therapeutic potential.
- 2012
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Ingår i: Future Oncology. - : Future Medicine Ltd. - 1479-6694 .- 1744-8301. ; 8:10, s. 1301-1313
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Tidskriftsartikel (refereegranskat)abstract
- Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.
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| 11. |
- Jaillon, Sébastien, et al.
(författare)
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The Humoral Pattern Recognition Molecule PTX3 Is a Key Component of Innate Immunity against Urinary Tract Infection.
- 2014
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Ingår i: Immunity. - : Elsevier BV. - 1074-7613. ; 40:4, s. 621-632
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Tidskriftsartikel (refereegranskat)abstract
- Immunity in the urinary tract has distinct and poorly understood pathophysiological characteristics and urinary tract infections (UTIs) are important causes of morbidity and mortality. We investigated the role of the soluble pattern recognition molecule pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, in UTIs. PTX3-deficient mice showed defective control of UTIs and exacerbated inflammation. Expression of PTX3 was induced in uroepithelial cells by uropathogenic Escherichia coli (UPEC) in a Toll-like receptor 4 (TLR4)- and MyD88-dependent manner. PTX3 enhanced UPEC phagocytosis and phagosome maturation by neutrophils. PTX3 was detected in urine of UTI patients and amounts correlated with disease severity. In cohorts of UTI-prone patients, PTX3 gene polymorphisms correlated with susceptibility to acute pyelonephritis and cystitis. These results suggest that PTX3 is an essential component of innate resistance against UTIs. Thus, the cellular and humoral arms of innate immunity exert complementary functions in mediating resistance against UTIs.
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| 12. |
- Lim, Chun Hwee, et al.
(författare)
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Thrombin-derived host defence peptide modulates neutrophil rolling and migration in vitro and functional response in vivo
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Host defence peptides (HDPs) derived from the C-terminus of thrombin are proteolytically generated by enzymes released during inflammation and wounding. In this work, we studied the effects of the prototypic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on neutrophil functions. In vitro, GKY25 was shown to decrease LPS-induced neutrophil activation. In addition, the peptide induced CD62L shedding on neutrophils without inducing their activation. Correspondingly, GKY25-treated neutrophils showed reduced attachment and rolling behaviour on surfaces coated with the CD62L ligand E-selectin. The GKY25-treated neutrophils also displayed a dampened chemotactic response against the chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited a reduced local ROS response against LPS. Taken together, our results show that GKY25 can modulate neutrophil functions in vitro and in vivo.
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| 13. |
- Liu, Yang, et al.
(författare)
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Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing
- 2023
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Ingår i: Acta Biomaterialia. - : Elsevier BV. - 1878-7568 .- 1742-7061. ; 162, s. 164-181
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Tidskriftsartikel (refereegranskat)abstract
- Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.
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| 14. |
- Lundgren, Sigrid, et al.
(författare)
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Analysis of bacteria, inflammation, and exudation in epidermal suction blister wounds reveals dynamic changes during wound healing
- 2023
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The skin microbiome undergoes dynamic changes during different phases of wound healing, however the role of bacteria in the wound healing process remains poorly described. In this study, we aimed to determine how wound bacteria develop over time in epidermal wounds, and how they interact with inflammatory processes during wound healing. To this end, we analyzed wound fluid and swab samples collected from epidermal suction blister wounds in healthy volunteers. We found that bacterial numbers, measured in swabs and dressing fluid, increased rapidly after wounding and stabilized by day 8. The composition of bacterial species identified by MALDI-TOF mass spectrometry differed between wounds, but generally consisted primarily of commensal bacteria and remained largely stable over time. Inflammation and neutrophil activity, measured by quantification of cytokines and neutrophil proteins in dressing fluid, peaked on day 5. Exudation, measured by quantification of protein content in dressings, also peaked at this time and strongly correlated with cytokine and neutrophil protein levels. Inflammation, neutrophil activity, and exudation were not correlated with bacterial counts at any time, indicating that in normally healing wounds, these processes are primarily driven by the host and are independent of colonizing bacteria. Our analysis provides a comprehensive understanding of epidermal wound healing dynamics in the host and the role of the microbiome in healthy wound healing.Competing Interest StatementA.S. is a founder of in2cure AB, a parent company of Xinnate AB which was the sponsor of the clinical trial from which the biobank samples used in this study are derived. G.P. is employed part-time (20%) by Xinnate AB. The other authors have declared that no conflict of interest exists.Clinical TrialNCT05378997Funding StatementThis study was funded by grants from the Swedish Research Council (project 2017-02341, 2020-02016), Edvard Welanders Stiftelse and Finsenstiftelsen (Hudfonden), the Royal Physiographic Society, the Crafoord and Österlund Foundations, and the Swedish Government Funds for Clinical Research (ALF).Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Swedish ethical review authority (etikprövningsmyndigheten) gave ethical approval for this work (application number 2022-00527-01).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesThe data that support the findings of this study are openly available in Zenodo at http://doi.org/10.5281/zenodo.10283373, reference number 10283373.BCAbicinchoninic acidCFUcolony forming unitsELISAenzyme-linked immunosorbent assayHBPheparin-binding proteinIFNinterferonILinterleukinMALDI-TOFMatrix Assisted Laser Desorption lonization -Time Of FlightMBTMALDI BiotyperMPOmyeloperoxidaseMSmass spectrometryNEneutrophil elastasePBSphosphate-buffered salineTCPthrombin-derived C-terminal peptideTNFtumor necrosis factor
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| 15. |
- Lutay, Nataliya, et al.
(författare)
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Bacterial control of host gene expression through RNA polymerase II
- 2013
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Ingår i: Journal of Clinical Investigation. - 0021-9738. ; 123:6, s. 2366-2379
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Tidskriftsartikel (refereegranskat)abstract
- The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coil develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II-dependent (Pol II-dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coil 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II-dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.
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| 16. |
- Markeviciute, Vetra, et al.
(författare)
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Systemically administered zoledronic acid activates locally implanted synthetic hydroxyapatite particles enhancing peri-implant bone formation : A regenerative medicine approach to improve fracture fixation
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Ingår i: Acta Biomaterialia. - 1742-7061.
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Tidskriftsartikel (refereegranskat)abstract
- Fracture fixation in an ageing population is challenging and fixation failure increases mortality and societal costs. We report a novel fracture fixation treatment by applying a hydroxyapatite (HA) based biomaterial at the bone-implant interface and biologically activating the biomaterial by systemic administration of a bisphosphonate (zoledronic acid, ZA). We first used an animal model of implant integration and applied a calcium sulphate (CaS)/HA biomaterial around a metallic screw in the tibia of osteoporotic rats. Using systemic ZA administration at 2-weeks post-surgery, we demonstrated that the implant surrounded by HA particles showed significantly higher peri‑implant bone formation compared to the unaugmented implants at 6-weeks. We then evaluated the optimal timing (day 1, 3, 7 and 14) of ZA administration to achieve a robust effect on peri‑implant bone formation. Using fluorescent ZA, we demonstrated that the uptake of ZA in the CaS/HA material was the highest at 3- and 7-days post-implantation and the uptake kinetics had a profound effect on the eventual peri‑implant bone formation. We furthered our concept in a feasibility study on trochanteric fracture patients randomized to either CaS/HA augmentation or no augmentation followed by systemic ZA treatment. Radiographically, the CaS/HA group showed signs of increased peri‑implant bone formation compared with the controls. Finally, apart from HA, we demonstrated that the concept of biologically activating a ceramic material by ZA could also be applied to β-tricalcium phosphate. This novel approach for fracture treatment that enhances immediate and long-term fracture fixation in osteoporotic bone could potentially reduce reoperations, morbidity and mortality. Statement of significance: • Fracture fixation in an ageing population is challenging. Biomaterial-based augmentation of fracture fixation devices has been attempted but lack of satisfactory biological response limits their widespread use. • We report the biological activation of locally implanted microparticulate hydroxyapatite (HA) particles placed around an implant by systemic administration of the bisphosphonate zoledronic acid (ZA). The biological activation of HA by ZA enhances peri‑implant bone formation. •Timing of ZA administration after HA implantation is critical for optimal ZA uptake and consequently determines the extent of peri‑implant bone formation. • We translate the developed concept from small animal models of implant integration to a proof-of-concept clinical study on osteoporotic trochanteric fracture patients. • ZA based biological activation can also be applied to other calcium phosphate biomaterials.
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| 17. |
- Menzel, Mandy, et al.
(författare)
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Caspase-1 deficiency reduces eosinophilia and interleukin-33 in an asthma exacerbation model
- 2017
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Ingår i: ERJ Open Research. - : European Respiratory Society (ERS). - 2312-0541. ; 3:4
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Tidskriftsartikel (refereegranskat)abstract
- Rhinovirus infections are common triggers of asthma exacerbations. Viruses can activate the inflammasome, resulting in processing and activation of caspase-1. This recruitment triggers production of interleukin (IL)-1β and IL-18, which have been implicated in asthma. Elucidating the involvement of the inflammasome and its compartments, such as caspase-1, in asthma exacerbations is warranted. Gene expression of caspase-1 was measured in rhinovirus-infected primary bronchial epithelial cells of asthmatic and healthy donors 24 h post-infection. In an in vivo exacerbation experiment C57BL/6 wild-type and caspase-1-/- mice were challenged with house dust mite followed by exposures to the viral mimic poly(I:C). General lung inflammatory parameters and levels of T-helper type 2 (Th2)-upstream cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 were assessed. Caspase-1 expression was elevated after rhinoviral infection exclusively in bronchial epithelial cells from asthmatics. In a translational mouse model of asthma exacerbation effects of caspase-1 on airway inflammation and Th2-upstream cytokines were explored. Caspase-1 deficient mice exhibited no alterations of general lung inflammatory parameters, but showed markedly reduced eosinophilia. Furthermore, the Th2-upstream cytokines IL-33, TSLP and IL-25 were reduced at exacerbation in mice lacking caspase-1. Rhinovirus infection increases bronchial epithelial caspase-1 in asthma. Caspase-1 may induce production of lung Th2-upstream cytokines and eosinophilia at exacerbations. Further targeting of caspase-1 signalling is warranted to explore its role in asthma exacerbations.
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| 18. |
- Menzel, Mandy, et al.
(författare)
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NFκB1 Dichotomously Regulates Pro-Inflammatory and Antiviral Responses in Asthma
- 2022
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 14:3, s. 182-191
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Tidskriftsartikel (refereegranskat)abstract
- Asthma exacerbations are commonly triggered by rhinovirus infections. Viruses can activate the NFκB pathway resulting in airway inflammation and increased Th2 cytokine expression. NFκB signaling is also involved in early activation of IFNβ, which is a central mediator of antiviral responses to rhinovirus infection. Using a mouse model, this study tests our hypothesis that NFκB signaling is involved in impaired IFNβ production at viral-induced asthma exacerbations. C57BL/6 wild-type and NFκB1 mice were challenged with house dust mite for 3 weeks and were subsequently stimulated with the rhinoviral mimic poly(I:C). General lung inflammatory parameters and levels of the Th2 upstream cytokine IL-33 were measured after allergen challenge. At exacerbation, production of IFNβ and antiviral proteins as well as gene expression of pattern recognition receptors and IRF3/IRF7 was assessed. In the asthma exacerbation mouse model, lack of NFκB1 resulted in lower levels of IL-33 after allergen challenge alone and was associated with reduced eosinophilia. At exacerbation, mice deficient in NFκB1 exhibited enhanced expression of IFNβ and antiviral proteins. This was accompanied by increased IRF3/IRF7 expression and induction of pattern recognition receptor expression. In a human asthma dataset, a negative correlation between IRF3 and NFκB1 expression was observed. NFκB may impair antiviral responses at exacerbation, possibly by reducing expression of the transcription factors IRF3/IRF7. These findings suggest a therapeutic potential for targeting NFκB pathways at viral infection-induced exacerbations.
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| 19. |
- Petrlova, Jitka, et al.
(författare)
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Selective protein aggregation confines and inhibits endotoxins in wounds : Linking host defense to amyloid formation
- 2023
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 26:10
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.
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| 20. |
- Petrlova, Jitka, et al.
(författare)
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Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products
- 2020
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 295:11, s. 3417-3430
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.
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| 21. |
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| 22. |
- Petruk, Ganna, et al.
(författare)
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SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity
- 2020
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Ingår i: Journal of Molecular Cell Biology. - : Oxford University Press (OUP). - 1759-4685. ; 12:12, s. 916-932
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Tidskriftsartikel (refereegranskat)abstract
- There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.
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| 23. |
- Petruk, Ganna, et al.
(författare)
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Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs
- 2023
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Ingår i: Nature Communications. - : Springer. - 2041-1723. ; 14, s. 1-20
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Tidskriftsartikel (refereegranskat)abstract
- There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
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| 24. |
- Petruk, Ganna, et al.
(författare)
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The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins
- 2021
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Ingår i: Journal of Lipid Research. - 0022-2275. ; 62
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Tidskriftsartikel (refereegranskat)abstract
- ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxin components such as lipopolysaccharide (LPS) and lipid A, yielding similar increases in the apoE α-helical content. Moreover, high-molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with lipid A. Together, our results demonstrate the ability of apoE to kill Gram-negative bacteria, interact with their endotoxins, which leads to the structural changes in apoE and the formation of aggregate-like complexes.
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| 25. |
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