Cell and Tissue Research

, Volume 379, Issue 1, pp 207–217 | Cite as

Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages

  • Karina M. Cirone
  • Priyoshi Lahiri
  • Ravi Holani
  • Yi Lin Tan
  • Rakel Arrazuria
  • Jeroen De Buck
  • Herman W. Barkema
  • Eduardo R. CoboEmail author
Regular Article


Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne’s disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1β cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.


Mycobacterium avium subsp. paratuberculosis Macrophages Cathelicidin LL-37 IL-8 



Immunofluorescence studies were conducted in the Live Cell Imaging Facility, Snyder Institute, University of Calgary.

Author contributions

KC acquired and analyzed all the data. PL and YT conducted the monocyte culture and qPCR data. RH conducted the macrophage imaging. RA created MAP A1-157 GFP, performed MAP culture and qPCR and live/dead assays. RA, JB, and HB provided critical scientific input. KC and EC conceived the experiment and wrote the manuscript.


This work was supported by the Margaret Gunn Endowment for Animal Research (UofC), NSERC Discovery (RGPAS-2017-507827) to EC and by the BEC.AR (Program for short term internship in biotechnology and agro-industry in BID country members sponsored by the Ministry of Education, Argentina) to KC.

Compliance with ethical statements

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

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Supplementary Figure 1

Confocal immune localization of MAP inside macrophages. GFP-MAP (K-10) (green) (MOI 5) was identified in murine macrophages at 3 h in comparison to control MOI 0. Macrophages were counterstained with phalloidin, which binds actin cytoskeleton (red) and DAPI as a nucleus maker (blue). Representative images for 3 independent experiments. Scale bar = 100 μm. (PNG 753 kb)

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High resolution image (TIFF 7357 kb)
441_2019_3098_Fig7_ESM.png (860 kb)
Supplementary Figure 2

The used doses of LL-37 are not cytotoxic in primary macrophages (a-b”) Visualization by optical microscopy of murine monocyte-derived (J774A.1) macrophages stimulated with LL-37 (2 μM; 1 h) and exposed to MAP (K-10) for up to 24 h. Scale bar = 60 μm. (c). The level of LDH release determined by ELISA from (J774A.1) macrophages treated with LL-37 (up to 50 μM; 1 h). Each column is an average of three individual LL-37 treatment. (PNG 859 kb)

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High resolution image (TIFF 6611 kb)
441_2019_3098_Fig8_ESM.png (195 kb)
Supplementary Figure 3

Expression of IL-1β, IL-10, β defensin-4 and CAMP on macrophages infected by MAP pre-treated with cathelicidins. Transcriptional gene expression of IL-1β (a-b), IL-10 (c-d) and β defensin-4 (e-f) was determined in MAP-infected macrophages pre-treated with LL-37 (2 μM; 1 h). mRNA expression was quantified with RT-qPCR. Means + SEM are shown. #p < 0.05 compared with MOI 0. (PNG 195 kb)

441_2019_3098_MOESM3_ESM.tiff (8.6 mb)
High resolution image (TIFF 8834 kb)


  1. Adams JL, Czuprynski CJ (1994) Mycobacterial cell wall components induce the production of TNF-alpha, IL-1, and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7. Microb Pathog 16:401–411PubMedGoogle Scholar
  2. Ahlstrom C, Barkema HW, Stevenson K, Zadoks RN, Biek R, Kao R, Trewby H, Haupstein D, Kelton DF, Fecteau G, Labrecque O, Keefe GP, McKenna SL, De Buck J (2015) Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level. BMC Genomics 16:161PubMedPubMedCentralGoogle Scholar
  3. Ahlstrom C, Barkema HW, De Buck J (2016a) Relative frequency of 4 major strain types of Mycobacterium avium ssp. paratuberculosis in Canadian dairy herds using a novel single nucleotide polymorphism-based polymerase chain reaction. J Dairy Sci 99:8297–8303PubMedGoogle Scholar
  4. Ahlstrom C, Barkema HW, Stevenson K, Zadoks RN, Biek R, Kao R, Trewby H, Haupstein D, Kelton DF, Fecteau G, Labrecque O, Keefe GP, McKenna SL, Tahlan K, De Buck J (2016b) Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle. PLoS One 11:e0149017PubMedPubMedCentralGoogle Scholar
  5. Alonso S, Pethe K, Russell DG, Purdy GE (2007) Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy. Proc Natl Acad Sci U S A 104:6031–6036PubMedPubMedCentralGoogle Scholar
  6. Anes E, Kuhnel MP, Bos E, Moniz-Pereira J, Habermann A, Griffiths G (2003) Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria. Nat Cell Biol 5:793–802PubMedGoogle Scholar
  7. Arsenault RJ, Maattanen P, Daigle J, Potter A, Griebel P, Napper S (2014) From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis. Vet Res 45:54–54PubMedPubMedCentralGoogle Scholar
  8. Bannantine JP, Stabel JR, Laws E, Cardieri MCD, Souza CD (2015) Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages. PLoS One 10:e0128966. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Barkema HW, Orsel K, Nielsen SS, Koets AP, Rutten V, Bannantine JP, Keefe GP, Kelton DF, Wells SJ, Whittington RJ, Mackintosh CG, Manning EJ, Weber MF, Heuer C, Forde TL, Ritter C, Roche S, Corbett CS, Wolf R, Griebel PJ, Kastelic JP, De Buck J (2018) Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection. Transbound Emerg Dis 65(Suppl 1):125–48PubMedGoogle Scholar
  10. Berns M, Hommes DW (2016) Anti-TNF-alpha therapies for the treatment of Crohn's disease: the past, present and future. Expert Opin Investig Drugs 25:129–143PubMedGoogle Scholar
  11. Boucher E, Marin M, Holani R, Young-Speirs M, Moore DM, Cobo ER (2018) Characteristic pro-inflammatory cytokines and host defence cathelicidin peptide produced by human monocyte-derived macrophages infected with Neospora caninum. Parasitology 145:871–884PubMedGoogle Scholar
  12. Burger E, Araujo A, Lopez-Yglesias A, Rajala MW, Geng L, Levine B, Hooper LV, Burstein E, Yarovinsky F (2018) Loss of Paneth cell autophagy causes acute susceptibility to Toxoplasma gondii-mediated inflammation. Cell Host Microbe 23:177–190 e174PubMedPubMedCentralGoogle Scholar
  13. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622PubMedPubMedCentralGoogle Scholar
  14. Chiodini RJ, Buergelt CD (1993) Susceptibility of Balb/c, C57/ B6 and C57/B10 mice to infection with Mycobacterium paratuberculosis. J Comp Pathol 109:309–319PubMedGoogle Scholar
  15. Cobo ER, Kissoon-Singh V, Moreau F, Chadee K (2015) Colonic MUC2 mucin regulates the expression and antimicrobial activity of beta-defensin 2. Mucosal Immunol 8:1360–1372PubMedPubMedCentralGoogle Scholar
  16. Cobo ER, Kissoon-Singh V, Moreau F, Holani R, Chadee K (2017) MUC2 mucin and butyrate contribute to the synthesis of the antimicrobial peptide cathelicidin in response to Entamoeba histolytica- and dextran sodium sulfate-induced colitis. Infect Immun 85:e00905-16.
  17. Colavecchia SB, Fernandez B, Jolly A, Minatel L, Hajos SE, Paolicchi FA, Mundo SL (2016) Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models. Vet Immunol Immunopathol 176:28–33PubMedGoogle Scholar
  18. Cooney MA, Steele JL, Steinberg H, Talaat AM (2014) A murine oral model for Mycobacterium avium subsp. paratuberculosis infection and immunomodulation with Lactobacillus casei ATCC 334. Front Cell Infect Microbiol 4:11.10.3389Google Scholar
  19. Corbett CS, Barkema HW, De Buck J (2018) Quantifying fecal shedding of Mycobacterium avium ssp. paratuberculosis from calves after experimental infection and exposure. J Dairy Sci 101:1478–1487PubMedGoogle Scholar
  20. Derakhshani H, De Buck J, Mortier R, Barkema HW, Krause DO, Khafipour E (2016) The features of fecal and ieal mucosa-associated microbiota in dairy calves during early infection with Mycobacterium avium subspecies paratuberculosis. Front Microbiol 7:426PubMedPubMedCentralGoogle Scholar
  21. Donnellan S, Stone V, Johnston H, Giardiello M, Owen A, Rannard S, Aljayyoussi G, Swift B, Tran L, Watkins C, Stevenson K (2017) Intracellular delivery of nano-formulated antituberculosis drugs enhances bactericidal activity. J Inter Nanomed 2:146–156Google Scholar
  22. Ferwerda G, Kullberg BJ, de Jong DJ, Girardin SE, Langenberg DM, van Crevel R, Ottenhoff TH, Van der Meer JW, Netea MG (2007) Mycobacterium paratuberculosis is recognized by Toll-like receptors and NOD2. J Leukoc Biol 82:1011–1018PubMedGoogle Scholar
  23. Fournier B (2013) The function of TLR2 during staphylococcal diseases. Front Cell Infect Microbiol 2:167PubMedPubMedCentralGoogle Scholar
  24. Gomes MS, Sousa Fernandes S, Cordeiro JV, Silva Gomes S, Vieira A, Appelberg R (2008) Engagement of Toll-like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non-oxidative and TNF-independent anti-mycobacterial activity. Eur J Immunol 38:2180–2189PubMedGoogle Scholar
  25. Hasan Z, Schlax C, Kuhn L, Lefkovits I, Young D, Thole J, Pieters J (1997) Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway. Mol Microbiol 24:545–553PubMedGoogle Scholar
  26. Hines ME, Stabel JR, Sweeney RW, Griffin F, Talaat AM, Bakker D, Benedictus G, Davis WC, de Lisle GW, Gardner IA, Juste RA, Kapur V, Koets A, McNair J, Pruitt G, Whitlock RH (2007) Experimental challenge models for Johne’s disease: a review and proposed international guidelines. Vet Microbiol 122:197–222PubMedGoogle Scholar
  27. Holani R, Marin M, Kastelic J, Cobo ER (2018) Host defense peptides as innate immunomodulators in the pathogenesis of colitis. In: Elsevier (ed) Antimicrobial peptides in gastrointestinal disease. Academic Press: Cambridge, MA, USA, pp 133–164Google Scholar
  28. Hostetter J, Steadham E, Haynes J, Bailey T, Cheville N (2003) Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells. Comp Immunol Microbiol Infect Dis 26:269–283PubMedGoogle Scholar
  29. Huntley JF, Stabel JR, Paustian ML, Reinhardt TA, Bannantine JP (2005) Expression library immunization confers pro-tection against Mycobacterium avium subsp. paratuberculosis infection. Infect Immun 73:6877–6884PubMedPubMedCentralGoogle Scholar
  30. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216PubMedPubMedCentralGoogle Scholar
  31. Koets AP, Eda S, Sreevatsan S (2015) The within host dynamics of Mycobacterium avium ssp. paratuberculosis infection in cattle: where time and place matter. Vet Res 46:61PubMedPubMedCentralGoogle Scholar
  32. Kuehnel MP, Goethe R, Habermann A, Mueller E, Rohde M, Griffiths G et al (2001) Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol 3:551–566PubMedGoogle Scholar
  33. Lamont EA, O'Grady SM, Davis WC, Eckstein T, Sreevatsan S (2012) Infection with Mycobacterium avium subsp. paratuberculosis results in rapid interleukin-1beta release and macrophage transepithelial migration. Infect Immun 80:3225–3235PubMedPubMedCentralGoogle Scholar
  34. Lamont EA, Xu WW, Sreevatsan S (2013) Host-Mycobacterium avium subsp. paratuberculosis interactome reveals a novel iron assimilation mechanism linked to nitric oxide stress during early infection. BMC Genomics 14:694PubMedPubMedCentralGoogle Scholar
  35. Lamont EA, Talaat AM, Coussens PM, Bannantine JP, Grohn YT, Katani R, Li LL, Kapur V, Sreevatsan S (2014) Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model. Front Cell Infect Microbiol 4:87PubMedPubMedCentralGoogle Scholar
  36. Lee SJ, Noh KT, Kang TH, Han HD, Shin SJ, Soh BY, Park JH, Shin YK, Kim HW, Yun CH, Park WS, Jung ID, Park YM (2014) The Mycobacterium avium subsp. paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4. BMB Rep 47:115–120PubMedPubMedCentralGoogle Scholar
  37. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL (2006) Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311:1770–1773PubMedGoogle Scholar
  38. Marin M, Holani R, Shah CB, Odeon A, Cobo ER (2017) Cathelicidin modulates synthesis of toll-like receptors (TLRs) 4 and 9 in colonic epithelium. Mol Immunol 91:249–258PubMedGoogle Scholar
  39. Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, Roche FM, Mu R, Doho GH, Pistolic J, Powers JP, Bryan J, Brinkman FS, Hancock RE (2006) Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 176:2455–2464PubMedGoogle Scholar
  40. Mortier RA, Barkema HW, De Buck J (2015) Susceptibility to and diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy calves: a review. Prev Vet Med 121:189–198PubMedGoogle Scholar
  41. Mucha R, Bhide MR, Chakurkar EB, Novak M, Mikula I Sr (2009) Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle. Vet Immunol Immunopathol 128:381–388PubMedGoogle Scholar
  42. Parker AE, Bermudez LE (1997) Expression of the green fluorescent protein (GFP) in Mycobacterium avium as a tool to study the interaction between mycobacteria and host cells. Microb Pathog 22:193–198PubMedGoogle Scholar
  43. Periasamy S, Tripathi BN, Singh N (2009) Mechanisms of Mycobacterium avium subsp. paratuberculosis induced apoptosis and necrosis in bovine macrophages. Vet Microbiol 165:392–401Google Scholar
  44. Quesniaux V, Fremond C, Jacobs M, Parida S, Nicolle D, Yeremeev V, Bihl F, Erard F, Botha T, Drennan M, Soler MN, Le Bert M, Schnyder B, Ryffel B (2004) Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect 6:946–959PubMedGoogle Scholar
  45. Ralph P, Prichard J, Cohn M (1975) Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. J Immunol 114:898–905PubMedGoogle Scholar
  46. Rode AKO, Kongsbak M, Hansen MM, Lopez DV, Levring TB, Woetmann A, Odum N, Bonefeld CM, Geisler C (2017) Vitamin D counteracts Mycobacterium tuberculosis-induced cathelicidin downregulation in dendritic cells and allows Th1 differentiation and IFNgamma secretion. Front Immunol 8:656PubMedPubMedCentralGoogle Scholar
  47. Rosseels V, Roupie V, Zinniel D, Barletta RG, Huygen K (2006) Development of luminescent Mycobacterium avium subsp. paratuberculosis for rapid screening of vaccine candidates in mice. Infect Immun 74:3684–3686PubMedPubMedCentralGoogle Scholar
  48. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017) ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:529PubMedPubMedCentralGoogle Scholar
  49. Santos JC, Silva-Gomes S, Silva JP, Gama M, Rosa G, Gallo RL, Appelberg R (2014) Endogenous cathelicidin production limits inflammation and protective immunity to Mycobacterium avium in mice. Immun Inflammation Dis 2:1–12Google Scholar
  50. Schaller-Bals S, Schulze A, Bals R (2002) Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med 165:992–995PubMedGoogle Scholar
  51. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675PubMedPubMedCentralGoogle Scholar
  52. Shin SJ, Wu C, Steinberg H, Talaat AM (2006) Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. Infect Immun 74:3825–3833PubMedPubMedCentralGoogle Scholar
  53. Slana I, Kralik P, Kralova A, Pavlik I (2008) On-farm spread of Mycobacterium avium ssp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol 128:250–257PubMedGoogle Scholar
  54. Sohal JS, Singh SV, Tyagi P, Subhodh S, Singh PK, Singh AV, Narayanasamy K, Sheoran N, Singh Sandhu K (2008) Immunology of mycobacterial infections: with special reference to Mycobacterium avium subspecies paratuberculosis. Immunobiology 213:585–598PubMedGoogle Scholar
  55. Sonawane A, Santos JC, Mishra BB, Jena P, Progida C, Sorensen OE, Gallo R, Appelberg R, Griffiths G (2011) Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages. Cell Microbiol 13:1601–1617PubMedGoogle Scholar
  56. Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678–681PubMedGoogle Scholar
  57. Tanaka S, Sato M, Taniguchi T, Yokomizo Y (1994) Histopathological and morphometrical comparison of granulomatous lesions in BALB/c and C3H/HeJ mice inoculated with Mycobacterium paratuberculosis. J Comp Pathol 110:381–388PubMedGoogle Scholar
  58. Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D, Deretic V (1998) Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 111(Pt 7):897–905PubMedGoogle Scholar
  59. Wang JJ, Chen C, Xie PF, Pan Y, Tan YH, Tang LJ (2014) Proteomic analysis and immune properties of exosomes released by macrophages infected with Mycobacterium avium. Microbes Infect 16:283–291PubMedGoogle Scholar
  60. Weiss DJ, Evanson OA, de Souza C, Abrahamsen MS (2005) A critical role of interleukin-10 in the response of bovine macrophages to infection by Mycobacterium avium subsp paratuberculosis. Am J Vet Res 66:721–726PubMedGoogle Scholar
  61. Weiss DJ, Souza CD, Evanson OA, Sanders M, Rutherford M (2008) Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium. J Leukoc Biol 83:48–55PubMedGoogle Scholar
  62. Yang CS, Shin DM, Kim KH, Lee ZW, Lee CH, Park SG, Bae YS, Jo EK (2009) NADPH oxidase 2 interaction with TLR2 is required for efficient innate immune responses to mycobacteria via cathelicidin expression. J Immunol 182:3696–3705PubMedGoogle Scholar
  63. Young-Speirs M, Drouin D, Cavalcante PA, Barkema HW, Cobo ER (2018) Host defense cathelicidins in cattle: types, production, bioactive functions and potential therapeutic and diagnostic applications. Int J Antimicrob AgentsGoogle Scholar
  64. Zanetti M (2005) The role of cathelicidins in the innate host defenses of mammals. Curr Issues Mol Biol 7:179–196PubMedGoogle Scholar
  65. Zur Lage S, Goethe R, Darji A, Valentin-Weigand P, Weiss S (2003) Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium. Immunology 108:62–69PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Animal ProductionNational Agricultural Technology InstituteBuenos AiresArgentina
  2. 2.Department of Production Animal Health, Faculty of Veterinary MedicineUniversity of CalgaryCalgaryCanada

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