| 1. |
- Hansen, Finja, et al.
(author)
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The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes
- 2015
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In: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 194:11, s. 5397-5406
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Journal article (peer-reviewed)abstract
- Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of human thrombin, inhibits proinflammatory responses in vitro and in vivo, but the mode of action is unclear. In this study, we show that GKY25, apart from binding bacterial LPS, also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo. Moreover, GKY25 inhibits TLR4-and TLR2-induced NF-kappa B activation in response to several microbe-derived agonists. Furthermore, GKY25 reduces LPS-induced phosphorylation of MAPKs p38 alpha and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25, based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-kappa B activity and proinflammatory cytokine production in monocytes and macrophages.
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| 2. |
- Kalle, Martina, et al.
(author)
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A Peptide of Heparin Cofactor II Inhibits Endotoxin-Mediated Shock and Invasive Pseudomonas aeruginosa Infection.
- 2014
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:7
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Journal article (peer-reviewed)abstract
- Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.
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| 3. |
- Kalle, Martina, et al.
(author)
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Host Defense Peptides of Thrombin Modulate Inflammation and Coagulation in Endotoxin-Mediated Shock and Pseudomonas aeruginosa Sepsis.
- 2012
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12
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Journal article (peer-reviewed)abstract
- Gram-negative sepsis is accompanied by a disproportionate innate immune response and excessive coagulation mainly induced by endotoxins released from bacteria. Due to rising antibiotic resistance and current lack of other effective treatments there is an urgent need for new therapies. We here present a new treatment concept for sepsis and endotoxin-mediated shock, based on host defense peptides from the C-terminal part of human thrombin, found to have a broad and inhibitory effect on multiple sepsis pathologies. Thus, the peptides abrogate pro-inflammatory cytokine responses to endotoxin in vitro and in vivo. Furthermore, they interfere with coagulation by modulating contact activation and tissue factor-mediated clotting in vitro, leading to normalization of coagulation responses in vivo, a previously unknown function of host defense peptides. In a mouse model of Pseudomonas aeruginosa sepsis, the peptide GKY25, while mediating a modest antimicrobial effect, significantly inhibited the pro-inflammatory response, decreased fibrin deposition and leakage in the lungs, as well as reduced mortality. Taken together, the capacity of such thrombin-derived peptides to simultaneously modulate bacterial levels, pro-inflammatory responses, and coagulation, renders them attractive therapeutic candidates for the treatment of invasive infections and sepsis.
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| 4. |
- Kalle, Martina, et al.
(author)
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Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule
- 2013
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 190:12, s. 6303-6310
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Journal article (peer-reviewed)abstract
- The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.
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| 5. |
- Kalle, Martina
(author)
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Targeting bacterial infections by using immunomodulatory host defence peptides and proteins
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Bacterial infections and sepsis are amongst the leading causes of death worldwide. Sepsis is caused by an uncontrolled systemic host response towards an invading pathogen. Due to the engagement of various systems including cellmediated responses, the coagulation and complement cascades, treatment of sepsis remains challenging. This is reflected in mortality rates of about 50 % for patients with septic shock, despite improved health care and antibiotic usage. Endogenous host defence peptides (HDPs) are essential components of the innate defence against invading pathogens. They exert multiple biological functions, and apart from their antimicrobial effects they may modulate inflammation, coagulation, and chemotaxis. Due to increasing bacterial resistance and due to the complex nature of severe bacterial infections, HDPs are today considered valuable candidates in the development of novel treatments for infections. In papers I-III the ability of HDPs, derived either from the C-terminus of human thrombin (HVF18, GKY25) or human tissue factor pathway inhibitor 2 (EDC34), to modulate innate immune responses caused by bacteria or lipopolysaccharide (LPS), were investigated. Thrombin-derived peptides significantly blocked LPS-induced responses including tissue-factor driven coagulation in vitro and in vivo and thereby improved survival in experimental animal models of LPS-induced shock and Pseudomonas aeruginosa sepsis (Paper I). The data in Paper II, demonstrate that the inhibition of these pro-inflammatory responses by GKY25, is not solely dependent on extracellular LPS-scavenging, but involves interactions with macrophages and monocytes. The TFPI-2 peptide (EDC34) also significantly modulated the coagulation cascade in vitro and in vivo and efficiently killed bacteria by enhancing complementmediated killing. The combination of these functions lead to improved survival in experimental models of E. coli or Pseudomonas sepsis (Paper III). In conclusion, the discovered immunomodulatory properties of these HDPs clearly indicate their potential for the development of new treatments for bacterial infections. Finally, a novel role of the abundant plasma protein heparin cofactor II (HCII) in host defence was discovered. HCII belongs to the class of serine proteinase inhibitors (serpins) and specifically inhibits human thrombin. However, its precise physiological role remained enigmatic. Paper IV shows that cleavage of HCII by human neutrophil elastase induces a conformational change in the HCII molecule, thereby uncovering a previously unknown antibacterial function of HCII.
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| 6. |
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| 7. |
- Karlsson, Max, et al.
(author)
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Genome-wide single cell annotation of the human protein-coding genes
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Other publication (other academic/artistic)abstract
- An important quest for the life science community is to deliver a complete annotation of the human building-blocks of life, the genes and the proteins. Here, we report on a genome-wide effort to annotate all protein-coding genes based on single cell transcriptomics data representing all major tissues and organs in the human body, integrated with data from bulk transcriptomics and antibody-based tissue profiling. Altogether, 25 tissues have been analyzed with single cell transcriptomics resulting in genome-wide expression in 444 single cell types using a strategy involving pooling data from individual cells to obtain genome-wide expression profiles of individual cell type. We introduce a new genome-wide classification tool based on clustering of similar expression profiles across single cell types, which can be visualized using dimensional reduction maps (UMAP). The clustering classification is integrated with a new “tau” score classification for all protein-coding genes, resulting in a measure of single cell specificity across all cell types for all individual genes. The analysis has allowed us to annotate all human protein-coding genes with regards to function and spatial distribution across individual cell types across all major tissues and organs in the human body. A new version of the open access Human Protein Atlas (www.proteinatlas.org) has been launched to enable researchers to explore the new genome-wide annotation on an individual gene level.
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| 8. |
- Kasetty, Gopinath, et al.
(author)
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Anti-endotoxic and antibacterial effects of a dermal substitute coated with host defense peptides.
- 2015
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In: Biomaterials. - : Elsevier BV. - 1878-5905 .- 0142-9612. ; 53, s. 415-425
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Journal article (peer-reviewed)abstract
- Biomaterials used during surgery and wound treatment are of increasing importance in modern medical care. In the present study we set out to evaluate the addition of thrombin-derived host defense peptides to human acellular dermis (hAD, i.e. epiflex(®)). Antimicrobial activity of the functionalized hAD was demonstrated using radial diffusion and viable count assays against Gram-negative Escherichia coli, Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Electron microscopy analyses showed that peptide-mediated bacterial killing led to reduced hAD degradation. Furthermore, peptide-functionalized hAD displayed endotoxin-binding activity in vitro, as evidenced by inhibition of NF-κB activation in human monocytic cells (THP-1 cells) and a reduction of pro-inflammatory cytokine production in whole blood in response to lipopolysaccharide stimulation. The dermal substitute retained its anti-endotoxic activity after washing, compatible with results showing that the hAD bound a significant amount of peptide. Furthermore, bacteria-induced contact activation was inhibited by peptide addition to the hAD. E. coli infected hAD, alone, or after treatment with the antiseptic substance polyhexamethylenebiguanide (PHMB), yielded NF-κB activation in THP-1 cells. The activation was abrogated by peptide addition. Thus, thrombin-derived HDPs should be of interest in the further development of new biomaterials with combined antimicrobial and anti-endotoxic functions for use in surgery and wound treatment.
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| 9. |
- Kasetty, Gopinath, et al.
(author)
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Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin
- 2011
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 55:6, s. 2880-2890
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Journal article (peer-reviewed)abstract
- Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length-and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (> 12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.
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| 10. |
- Kasetty, Gopinath, et al.
(author)
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The C-Terminal Sequence of Several Human Serine Proteases Encodes Host Defense Functions.
- 2011
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In: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 3:5, s. 471-482
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Journal article (peer-reviewed)abstract
- Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest.
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