Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv

Los neutrófilos son células de la inmunidad innata que predominan en pacientes con tuberculosis activa, estos polimorfonucleares mediante la apoptosis, pueden contribuir en la resolución de la infección sin ocasionar daño en el tejido pulmonar, favorecer la presentación antigénica e inducir la produ...

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Autores:
Alfonso Alfonso, Angie Paola
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2019
Institución:
Colegio Mayor de Cundinamarca
Repositorio:
Repositorio Colegio Mayor de Cundinamarca
Idioma:
spa
OAI Identifier:
oai:repositorio.unicolmayor.edu.co:unicolmayor/307
Acceso en línea:
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/307
Palabra clave:
Enfermedades micobacterianas
Epidemiología
Tuberculosis pulmonar
Tuberculosis
Mycobacterium tuberculosis
Neutrófilos
Anticuerpos
Apoptosis.
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openAccess
License
Derechos Reservados -Universidad Colegio Myor de Cundinamarca ,2019
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network_name_str Repositorio Colegio Mayor de Cundinamarca
repository_id_str
dc.title.spa.fl_str_mv Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
title Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
spellingShingle Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
Enfermedades micobacterianas
Epidemiología
Tuberculosis pulmonar
Tuberculosis
Mycobacterium tuberculosis
Neutrófilos
Anticuerpos
Apoptosis.
title_short Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
title_full Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
title_fullStr Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
title_full_unstemmed Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
title_sort Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rv
dc.creator.fl_str_mv Alfonso Alfonso, Angie Paola
dc.contributor.advisor.none.fl_str_mv Carabalí Isajar, Mary Lilián
Castro Molina, Susan Lorena
dc.contributor.author.none.fl_str_mv Alfonso Alfonso, Angie Paola
dc.contributor.corporatename.spa.fl_str_mv Universidad Colegio Mayor de Cundinamarca
dc.contributor.researchgroup.spa.fl_str_mv Trabajo de grado
dc.subject.lemb.none.fl_str_mv Enfermedades micobacterianas
Epidemiología
Tuberculosis pulmonar
topic Enfermedades micobacterianas
Epidemiología
Tuberculosis pulmonar
Tuberculosis
Mycobacterium tuberculosis
Neutrófilos
Anticuerpos
Apoptosis.
dc.subject.proposal.spa.fl_str_mv Tuberculosis
Mycobacterium tuberculosis
Neutrófilos
Anticuerpos
Apoptosis.
description Los neutrófilos son células de la inmunidad innata que predominan en pacientes con tuberculosis activa, estos polimorfonucleares mediante la apoptosis, pueden contribuir en la resolución de la infección sin ocasionar daño en el tejido pulmonar, favorecer la presentación antigénica e inducir la producción de citoquinas proinflamatorias, para contrarrestar el crecimiento micobacteriano; sin embargo, los factores de patogenicidad y virulencia que posee Mycobacterium tuberculosis, pueden producir otras formas de muerte celular que ocasionan daño tisular y favorecen la inflamación exacerbada. La apoptosis puede ser inducida por fagocitosis que se lleva a cabo cuando los antígenos se encuentran unidos a anticuerpos (opsonofagocitosis), este fenómeno no se ha descrito en procesos infecciosos relacionados con Mycobacterium y considerando la importancia de este tipo de muerte celular como parte de una respuesta inmune protectiva, es necesario evaluar anticuerpos que promuevan este tipo de muerte progamada. En este sentido, se planteó el siguiente trabajo, evaluar si la opsonofagocitosis con IgGs que reconocen antígenos de la micobacteria, puede mediar la apoptosis de neutrófilos, por consiguiente, se seleccionaron y aislaron inmunoglobulinas G, que reconocieron 9 secuencias peptídicas presentes en diferentes proteínas de la envoltura de Mycobacterium tuberculosis H37Rv. Se encontró que la opsonofagocitosis con los anticuerpos péptidos-específicos 37964, 37765, 31107, 16300 y 38373 aumentaron la apoptosis en los polimorfonucleares, lo cual podría indicar que algunas inmunoglobulinas, posiblemente pueden mediar la apoptosis en estas células, de forma que los péptidos que son reconocidos y para los cuales se aislaron las IgGs, probablemente pueden favorecer de alguna forma la generación de una respuesta inmune protectiva.
publishDate 2019
dc.date.issued.none.fl_str_mv 2019-11
dc.date.accessioned.none.fl_str_mv 2021-06-30T15:02:04Z
dc.date.available.none.fl_str_mv 2021-06-30T15:02:04Z
dc.type.spa.fl_str_mv Trabajo de grado - Pregrado
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dc.relation.references.spa.fl_str_mv 1. Bozzano F, Marras F and De Maria A. Immunology of tuberculosis. Mediterr J Hematol Infect Dis. 2014; 6.
2. WHO. World Health Organization: Global Tuberculosis report 2018.
3. INS. Instituto Nacional de Salud: Boletín epidemiológico semana 11 del 2019.
4. Garcia J, Puentes A, Rodriguez L, et al. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines. Protein Sci. 2005; 14: 2236-45.
5. Forero M, Puentes A, Cortes J, et al. Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells. Protein Sci. 2005; 14: 2767-80.
6. Vera-Bravo R, Torres E, Valbuena JJ, et al. Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines. Biochem Biophys Res Commun. 2005; 332: 771-81.
7. Patarroyo MA, Curtidor H, Plaza DF, et al. Peptides derived from the Mycobacterium tuberculosis Rv1490 surface protein implicated in inhibition of epithelial cell entry: potential vaccine candidates? Vaccine. 2008; 26: 4387-95.
8. Plaza DF, Curtidor H, Patarroyo MA, et al. The Mycobacterium tuberculosis membrane protein Rv2560--biochemical and functional studies. FEBS J. 2007; 274: 6352-64.
9. Patarroyo MA, Plaza DF, Ocampo M, et al. Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein. Biochem Biophys Res Commun. 2008; 372: 935-40
10. Cifuentes DP, Ocampo M, Curtidor H, et al. Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: potential TB vaccine candidate antigen. BMC Microbiol. 2010; 10: 109.
11. Caceres SM, Ocampo M, Arevalo-Pinzon G, Jimenez RA, Patarroyo ME and Patarroyo MA. The Mycobacterium tuberculosis membrane protein Rv0180c: Evaluation of peptide sequences implicated in mycobacterial invasion of two human cell lines. Peptides. 2011; 32: 1- 10.
12. Ocampo M, Aristizábal-Ramírez D, Rodríguez DM, et al. The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines. Protein Engineering, Design and Selection. 2012; 25: 235-42.
13. Ocampo M, Rodriguez DM, Curtidor H, Vanegas M, Patarroyo MA and Patarroyo ME. Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages. Amino Acids. 2012; 42: 2067-77.
14. Ocampo M, Rodriguez DC, Rodriguez J, et al. Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells. Bioorg Med Chem. 2013; 21: 6650-6.
15. Rodriguez DC, Ocampo M, Varela Y, Curtidor H, Patarroyo MA and Patarroyo ME. Mce4F Mycobacterium tuberculosis protein peptides can inhibit invasion of human cell lines. Pathog Dis. 2015; 73.
16. Rodríguez DM, Ocampo M, Curtidor H, Vanegas M, Patarroyo ME and Patarroyo MA. Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion. Biol Chem. 2012; 38: 208–16.
17. Rodriguez DC, Ocampo M, Reyes C, et al. Cell-Peptide Specific Interaction Can Inhibit Mycobacterium tuberculosis H37Rv Infection. J Cell Biochem. 2016; 117: 946-58.
18. Diaz DP, Ocampo M, Varela Y, Curtidor H, Patarroyo MA and Patarroyo ME. Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion. Mol Cell Biochem. 2017; 430: 149-60.
19. Ocampo M, Curtidor H, Vanegas M, Patarroyo MA and Patarroyo ME. Specific interaction between Mycobacterium tuberculosis lipoprotein-derived peptides and target cells inhibits mycobacterial entry in vitro. Chem Biol Drug Des. 2014; 84: 626-41.
20. Rodríguez DM, Vizcaíno C, Ocampo M, et al. Peptides from the Mycobacterium tuberculosis Rv1980c protein involved in human cell infection: insights into new synthetic subunit vaccine candidates. Biol Chem. 2010; 391: 207-2017.
21. Sanchez-Barinas CD, Ocampo M, Vanegas M, Castaneda-Ramirez JJ, Patarroyo MA and Patarroyo ME. Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions. Molecules. 2018; 23.
22. Chapeton-Montes JA, Plaza DF, Curtidor H, et al. Characterizing the Mycobacterium tuberculosis Rv2707 protein and determining its sequences which specifically bind to two human cell lines. Protein Sci. 2008; 17: 342–51.
23. Carabali-Isajar ML, Ocampo M, Rodriguez DC, et al. Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells. Bioorg Med Chem. 2018; 26: 2401-9.
24. Dallenga T and Schaible UE. Neutrophils in tuberculosis--first line of defence or booster of disease and targets for host-directed therapy? Pathog Dis. 2016; 74.
25. Warren E, Teskey G and Venketaraman V. Effector Mechanisms of Neutrophils within the Innate Immune System in Response to Mycobacterium tuberculosis Infection. J Clin Med. 2017; 6.
26. Lyadova IV. Neutrophils in Tuberculosis: Heterogeneity Shapes the Way? Mediators Inflamm. 2017; 2017: 8619307.
27. Quinn TM, DeLeo, F.R. Neutrophil Methods and Protocols. Humana Press 2da ed. 2014.
28. Geering B and Simon HU. Peculiarities of cell death mechanisms in neutrophils. Cell Death Differ. 2011; 18: 1457-69.
29. Dallenga T, Repnik U, Corleis B, et al. M. tuberculosis-Induced Necrosis of Infected Neutrophils Promotes Bacterial Growth Following Phagocytosis by Macrophages. Cell Host Microbe. 2017; 22: 519-30 e3.
30. Braian C, Hogea V and Stendahl O. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages. J Innate Immun. 2013; 5: 591-602.
31. Ramos-Kichik V, Mondragon-Flores R, Mondragon-Castelan M, et al. Neutrophil extracellular traps are induced by Mycobacterium tuberculosis. Tuberculosis (Edinb). 2009; 89: 29-37.
32. Gamberale R, Giordano M, Trevani AS, Andonegui G and Geffner JR. Modulation of human neutrophil apoptosis by immune complexes. J Immunol. 1998; 161: 3666-74.
33. Lu LL, Chung AW, Rosebrock TR, et al. A Functional Role for Antibodies in Tuberculosis. Cell. 2016; 167: 433-43
34. Lu LL, Suscovich TJ, Fortune SM and Alter G. Beyond binding: antibody effector functions in infectious diseases. Nat Rev Immunol. 2017; 18: 46-61.
35. Farga V and Caminero JA. Tuberculosis. . Mediterraneo 3ra ed. 2011.
36. Kaufmann SH. How can immunology contribute to the control of tuberculosis? Nat Rev Immunol. 2001; 1: 20-30.
37. Bozzano F, Marras F and De Maria A. Immunology of tuberculosis. Mediterr J Hematol Infect Dis. 2014; 6: e2014027.
38. Corleis B, Korbel D, Wilson R, Bylund J, Chee R and Schaible UE. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils. Cell Microbiol. 2012; 14: 1109-21.
39. Hilda NJ and Das S. Neutrophil CD64, TLR2 and TLR4 expression increases but phagocytic potential decreases during tuberculosis. Tuberculosis (Edinb). 2018; 111: 135-42.
40. Aleyd E, van Hout MW, Ganzevles SH, et al. IgA enhances NETosis and release of neutrophil extracellular traps by polymorphonuclear cells via Fcalpha receptor I. J Immunol. 2014; 192: 2374-83.
41. Mendez-Samperio P. Commentary: The Role of Neutrophils in the Induction of Specific Th1 and Th17 during Vaccination against Tuberculosis. Front Cell Infect Microbiol. 2017; 7: 179.
42. Barberis I, Bragazzi NL, Galluzzo L and Martini M. The history of tuberculosis: from the first historical records to the isolation of Koch's bacillus. J Prev Med Hyg. 2017; 58: E9-E12.
43. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ and Berry MP. The immune response in tuberculosis. Annu Rev Immunol. 2013; 31: 475-527.
44. Kumar K and Kon OM. Diagnosis and treatment of tuberculosis: latest developments and future priorities. Annals of Research Hospitals. 2017; 1.
45. Sua LF, Fernández, L. Tuberculosis miliar o diseminada. Revista Colombiana de Neumología. 2015; 27: 208 - 10.
46. Kleinnijenhuis J, Oosting M, Joosten LA, Netea MG and Van Crevel R. Innate immune recognition of Mycobacterium tuberculosis. Clin Dev Immunol. 2011; 2011: 405310.
47. Forrellad MA, Klepp LI, Gioffre A, et al. Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013; 4: 3-66.
48. INS. Instituto Nacional de Salud. Tuberculosis: Protocolos de vigilancia y salud publica 2016.
49. INS. Instituto Nacional de Salud. Guía para la vigilancia por laboratorio de tuberculosis. 2017.
50. OPS. Organización Panamericana de Salud. MANUAL para el diagnóstico bacteriológico de la tuberculosis 2008.
51. Pai M, Denkinger CM, Kik SV, et al. Gamma interferon release assays for detection of Mycobacterium tuberculosis infection. Clin Microbiol Rev. 2014; 27: 3-20.
52. Garcia R, Lado FL, Bastida V, Pérez ML and Ortiz A. Tratamiento actual de la tuberculosis. An Med Interna. 2003; 20: 91-100.
53. Li W, Deng G, Li M, Liu X and Wang Y. Roles of Mucosal Immunity against Mycobacterium tuberculosis Infection. Tuberc Res Treat. 2012; 2012: 791728.
54. Liu CH, Liu H and Ge B. Innate immunity in tuberculosis: host defense vs pathogen evasion. Cell Mol Immunol. 2017; 14: 963-75.
55. Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol. 2012; 12: 352-66. 56. Scordo JM, Knoell DL and Torrelles JB. Alveolar Epithelial Cells in Mycobacterium tuberculosis Infection: Active Players or Innocent Bystanders? J Innate Immun. 2016; 8: 3-14.
57. Lerner TR, Borel S and Gutierrez MG. The innate immune response in human tuberculosis. Cell Microbiol. 2015; 17: 1277-85.
58. McClean CM and Tobin DM. Macrophage form, function, and phenotype in mycobacterial infection: lessons from tuberculosis and other diseases. Pathog Dis. 2016; 74.
59. Mihret A. The role of dendritic cells in Mycobacterium tuberculosis infection. Virulence. 2012; 3: 654-9.
60. Allen M, Bailey C, Cahatol I, et al. Mechanisms of Control of Mycobacterium tuberculosis by NK Cells: Role of Glutathione. Front Immunol. 2015; 6: 508.
61. Eruslanov EB, Lyadova IV, Kondratieva TK, et al. Neutrophil Responses to Mycobacterium tuberculosis Infection in Genetically Susceptible and Resistant Mice. Infection and Immunity. 2005; 73: 1744-53.
62. Cummings BS and Schnellmann RG. Measurement of cell death in mammalian cells. Curr Protoc Pharmacol. 2004; Chapter 12: Unit 12 8.
63. Driss EK, János, G.F. Modulation of neutrophil apoptosis and the resolution of inflammation through β2 integrins. Front Immunol. 2013; 4: 1-15.
64. Tan BH, Meinken C, Bastian M, et al. Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens. J Immunol. 2006; 177: 1864-71
65. Andersson H, Andersson B, Eklund D, et al. Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages. PLoS One. 2014; 9.
66. Dheda K, Schwander SK, Zhu B, van Zyl-Smit RN and Zhang Y. The immunology of tuberculosis: from bench to bedside. Respirology. 2010; 15: 433-50.
67. Lin PL and Flynn JL. CD8 T cells and Mycobacterium tuberculosis infection. Semin Immunopathol. 2015; 37: 239-49.
68. Kozakiewicz L, Phuah J, Flynn J and Chan J. The role of B cells and humoral immunity in Mycobacterium tuberculosis infection. Adv Exp Med Biol. 2013; 783: 225-50.
69. Abebe F and Bjune G. The protective role of antibody responses during Mycobacterium tuberculosis infection. Clin Exp Immunol. 2009; 157: 235-43.
70. Rao M, Valentini D, Poiret T, et al. B in TB: B Cells as Mediators of Clinically Relevant Immune Responses in Tuberculosis. Clin Infect Dis. 2015; 61Suppl 3: S225-34.
71. Ocampo M, Patarroyo MA, Vanegas M, Alba MP and Patarroyo ME. Functional, biochemical and 3D studies of Mycobacterium tuberculosis protein peptides for an effective anti-tuberculosis vaccine. Crit Rev Microbiol. 2014; 40: 117-45.
72. The enzyme-linked immunosorbent assay (ELISA). Bulletin of the World Health Organization. 1976; 54: 129-39.
73. Merrifield R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a tetrapeptide Journal of the American Chemical Society. 1963; 85: 2149-54.
74. Houghten RA. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA. 1985; 82: 5131-5.
75. Amersham Biosciences Limited. CNBr-activated Sepharose 4B. 2002, p. 1-12.
76. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by co
77. Andersson AM, Larsson M, Stendahl O and Blomgran R. Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner. J Innate Immun. 2019: 1-13.
78. Larsen MH, Biermann, K, Jacobs, W.R. Laboratory Maintenance of Mycobacterium tuberculosis. Current Protocols in Microbiology. 2017; 6: 10A. 1. 1-A. 8.1.
79. Perskvist N, Long M, Stendahl O and Zheng L. Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway. J Immunol. 2002; 168: 6358-65.
80. Eum SY, Kong JH, Hong MS, et al. Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB. Chest. 2010; 137: 122
81. Blomgran R, Desvignes L, Briken V and Ernst JD. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells. Cell Host Microbe. 2012; 11: 81-90.
82. Kroon EE, Coussens AK, Kinnear C, et al. Neutrophils: Innate Effectors of TB Resistance? Front Immunol. 2018; 9: 2637.
83. Lowe DM, Redford PS, Wilkinson RJ, O'Garra A and Martineau AR. Neutrophils in tuberculosis: friend or foe? Trends Immunol. 2012; 33: 14-25.
84. Velmurugan K, Chen B, Miller JL, et al. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog. 2007; 3: e110.
85. Lu LL, Smith MT, Yu KKQ, et al. IFN-gamma-independent immune markers of Mycobacterium tuberculosis exposure. Nat Med. 2019; 25: 977-87.
86. Li H, Wang, X.X, Wang B, et al. Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis. Proc Natl Acad Sci 2017: 5023--8
87. Lu LL, Smith, M.T., Yu KKQ3, Luedemann C2, Suscovich TJ2, Grace PS2, Cain A2, Yu WH2,4, McKitrick TR5, Lauffenburger D4, Cummings RD5, Mayanja-Kizza H6, Hawn TR3, Boom WH7, Stein CM7,8, Fortune SM1,2, Seshadri C9, Alter G10. IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure Nat Med. 2019; 25: 977-87.
88. Zimmermann N, Thormann V, Hu B, et al. Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis. EMBO Mol Med. 2016; 8: 1325-39.
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spelling Carabalí Isajar, Mary Lilián2b4923d54898251e7b414131eb53be9fCastro Molina, Susan Lorena0538d7d5bb55e1323777cf3c55bf6d1aAlfonso Alfonso, Angie Paolaef2a72c82f084b30f61237e30915d3bcUniversidad Colegio Mayor de CundinamarcaTrabajo de grado2021-06-30T15:02:04Z2021-06-30T15:02:04Z2019-11https://repositorio.unicolmayor.edu.co/handle/unicolmayor/30760186Los neutrófilos son células de la inmunidad innata que predominan en pacientes con tuberculosis activa, estos polimorfonucleares mediante la apoptosis, pueden contribuir en la resolución de la infección sin ocasionar daño en el tejido pulmonar, favorecer la presentación antigénica e inducir la producción de citoquinas proinflamatorias, para contrarrestar el crecimiento micobacteriano; sin embargo, los factores de patogenicidad y virulencia que posee Mycobacterium tuberculosis, pueden producir otras formas de muerte celular que ocasionan daño tisular y favorecen la inflamación exacerbada. La apoptosis puede ser inducida por fagocitosis que se lleva a cabo cuando los antígenos se encuentran unidos a anticuerpos (opsonofagocitosis), este fenómeno no se ha descrito en procesos infecciosos relacionados con Mycobacterium y considerando la importancia de este tipo de muerte celular como parte de una respuesta inmune protectiva, es necesario evaluar anticuerpos que promuevan este tipo de muerte progamada. En este sentido, se planteó el siguiente trabajo, evaluar si la opsonofagocitosis con IgGs que reconocen antígenos de la micobacteria, puede mediar la apoptosis de neutrófilos, por consiguiente, se seleccionaron y aislaron inmunoglobulinas G, que reconocieron 9 secuencias peptídicas presentes en diferentes proteínas de la envoltura de Mycobacterium tuberculosis H37Rv. Se encontró que la opsonofagocitosis con los anticuerpos péptidos-específicos 37964, 37765, 31107, 16300 y 38373 aumentaron la apoptosis en los polimorfonucleares, lo cual podría indicar que algunas inmunoglobulinas, posiblemente pueden mediar la apoptosis en estas células, de forma que los péptidos que son reconocidos y para los cuales se aislaron las IgGs, probablemente pueden favorecer de alguna forma la generación de una respuesta inmune protectiva.1. Resumen 13 2. Introducción 14 3. Objetivos 16 3.1. Objetivo general 16 3.2. Objetivos específicos 16 4. Antecedentes 17 5. Marco Teórico 19 5.1. Generalidades de la tuberculosis 19 5.2. Mycobacterium tuberculosis y el complejo MTB 20 5.3. Epidemiología de la tuberculosis 21 5.4. Diagnóstico de la tuberculosis 23 5.4.1. Criterio bacteriológico 24 5.4.2. Criterio histopatológico 24 5.4.3. Criterio tuberculínico y prueba QuantiFERON 25 5.5. Tratamiento para la TB 25 5.6. Respuesta inmune innata ante Mycobacterium tuberculosis 26 5.6.1. Células epiteliales alveolares 27 5.6.2. Macrófagos 28 5.6.3. Células dendríticas (DCs) 29 5.6.4. Células asesinas naturales (NKs) 29 5.6.5. Neutrófilos 30 5.7. Respuesta inmune adaptativa ante Mycobacterium tuberculosis 31 5.7.1. Linfocitos T (LT) 31 5.7.2. Linfocitos B (LB) 32 5.8. Desarrollo de vacunas contra la tuberculosis 33 6. Metodología 35 6.1. Diseño metodológico 35 6.1.1. Tipo de investigación 35 6.1.2. Universo 35 6.1.3. Población 35 6.1.4. Muestra 35 6.2. Técnicas y procedimientos 36 6.2.1. Selección de péptidos 36 6.2.1.1. Aproximación bioinformática 36 6.2.1.2. Aproximación experimental 37 6.2.1.2.1. Obtención de sueros de Donantes 37 6.2.1.2.2. Búsqueda de anticuerpos IgGs afines a péptidos con epítopes B 37 6.2.2. Obtención de plasmas para purificación de inmunoglobulina G que reconocen péptidos seleccionados 38 6.2.2.1. Purificación de IgG 38 6.2.2.2. Purificación de IgG péptido-específica 38 6.2.3. Ensayos de fagocitosis en neutrófilos mediada por anticuerpos 39 6.2.3.1. Aislamiento de neutrófilos de sangre periférica 39 6.2.3.2. Ensayo de opsonofagocitosis 40 6.2.3.2.1. Cultivo de Mtb H37Rv 40 6.2.3.2.2. Opsonofagocitosis de Mtb H37Rv 40 6.2.4. Procesamiento y análisis de datos 41 7. Resultados 42 7.1. Selección de péptidos provenientes de proteínas de la envoltura de Mtb H37Rv 42 7.2. Purificación de inmunoglobulina G que reconocen péptidos específicos 45 7.3. Apoptosis de neutrófilos 47 8. Discusión 53 9. Conclusiones 57 10. Recomendaciones 58 11. Referencias 59 12. Anexos 66PregradoBacteriólogo(a) y Laboratorista ClínicoTrabajo de grado67p.application/pdfspaUniversidad Colegio Mayor de CundinamarcaFacultad de Ciencias de la SaludBogotá, Distrito CapitalBacteriología y Laboratorio ClínicoNo objeto asociado1. Bozzano F, Marras F and De Maria A. Immunology of tuberculosis. Mediterr J Hematol Infect Dis. 2014; 6.2. WHO. World Health Organization: Global Tuberculosis report 2018.3. INS. Instituto Nacional de Salud: Boletín epidemiológico semana 11 del 2019.4. Garcia J, Puentes A, Rodriguez L, et al. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines. Protein Sci. 2005; 14: 2236-45.5. Forero M, Puentes A, Cortes J, et al. Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells. Protein Sci. 2005; 14: 2767-80.6. Vera-Bravo R, Torres E, Valbuena JJ, et al. Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines. Biochem Biophys Res Commun. 2005; 332: 771-81.7. Patarroyo MA, Curtidor H, Plaza DF, et al. Peptides derived from the Mycobacterium tuberculosis Rv1490 surface protein implicated in inhibition of epithelial cell entry: potential vaccine candidates? Vaccine. 2008; 26: 4387-95.8. Plaza DF, Curtidor H, Patarroyo MA, et al. The Mycobacterium tuberculosis membrane protein Rv2560--biochemical and functional studies. FEBS J. 2007; 274: 6352-64.9. Patarroyo MA, Plaza DF, Ocampo M, et al. Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein. Biochem Biophys Res Commun. 2008; 372: 935-4010. Cifuentes DP, Ocampo M, Curtidor H, et al. Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: potential TB vaccine candidate antigen. BMC Microbiol. 2010; 10: 109.11. Caceres SM, Ocampo M, Arevalo-Pinzon G, Jimenez RA, Patarroyo ME and Patarroyo MA. The Mycobacterium tuberculosis membrane protein Rv0180c: Evaluation of peptide sequences implicated in mycobacterial invasion of two human cell lines. Peptides. 2011; 32: 1- 10.12. Ocampo M, Aristizábal-Ramírez D, Rodríguez DM, et al. The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines. Protein Engineering, Design and Selection. 2012; 25: 235-42.13. Ocampo M, Rodriguez DM, Curtidor H, Vanegas M, Patarroyo MA and Patarroyo ME. Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages. Amino Acids. 2012; 42: 2067-77.14. Ocampo M, Rodriguez DC, Rodriguez J, et al. Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells. Bioorg Med Chem. 2013; 21: 6650-6.15. Rodriguez DC, Ocampo M, Varela Y, Curtidor H, Patarroyo MA and Patarroyo ME. Mce4F Mycobacterium tuberculosis protein peptides can inhibit invasion of human cell lines. Pathog Dis. 2015; 73.16. Rodríguez DM, Ocampo M, Curtidor H, Vanegas M, Patarroyo ME and Patarroyo MA. Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion. Biol Chem. 2012; 38: 208–16.17. Rodriguez DC, Ocampo M, Reyes C, et al. Cell-Peptide Specific Interaction Can Inhibit Mycobacterium tuberculosis H37Rv Infection. J Cell Biochem. 2016; 117: 946-58.18. Diaz DP, Ocampo M, Varela Y, Curtidor H, Patarroyo MA and Patarroyo ME. Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion. Mol Cell Biochem. 2017; 430: 149-60.19. Ocampo M, Curtidor H, Vanegas M, Patarroyo MA and Patarroyo ME. Specific interaction between Mycobacterium tuberculosis lipoprotein-derived peptides and target cells inhibits mycobacterial entry in vitro. Chem Biol Drug Des. 2014; 84: 626-41.20. Rodríguez DM, Vizcaíno C, Ocampo M, et al. Peptides from the Mycobacterium tuberculosis Rv1980c protein involved in human cell infection: insights into new synthetic subunit vaccine candidates. Biol Chem. 2010; 391: 207-2017.21. Sanchez-Barinas CD, Ocampo M, Vanegas M, Castaneda-Ramirez JJ, Patarroyo MA and Patarroyo ME. Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions. Molecules. 2018; 23.22. Chapeton-Montes JA, Plaza DF, Curtidor H, et al. Characterizing the Mycobacterium tuberculosis Rv2707 protein and determining its sequences which specifically bind to two human cell lines. Protein Sci. 2008; 17: 342–51.23. Carabali-Isajar ML, Ocampo M, Rodriguez DC, et al. Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells. Bioorg Med Chem. 2018; 26: 2401-9.24. Dallenga T and Schaible UE. Neutrophils in tuberculosis--first line of defence or booster of disease and targets for host-directed therapy? Pathog Dis. 2016; 74.25. Warren E, Teskey G and Venketaraman V. Effector Mechanisms of Neutrophils within the Innate Immune System in Response to Mycobacterium tuberculosis Infection. J Clin Med. 2017; 6.26. Lyadova IV. Neutrophils in Tuberculosis: Heterogeneity Shapes the Way? Mediators Inflamm. 2017; 2017: 8619307.27. Quinn TM, DeLeo, F.R. Neutrophil Methods and Protocols. Humana Press 2da ed. 2014.28. Geering B and Simon HU. Peculiarities of cell death mechanisms in neutrophils. Cell Death Differ. 2011; 18: 1457-69.29. Dallenga T, Repnik U, Corleis B, et al. M. tuberculosis-Induced Necrosis of Infected Neutrophils Promotes Bacterial Growth Following Phagocytosis by Macrophages. Cell Host Microbe. 2017; 22: 519-30 e3.30. Braian C, Hogea V and Stendahl O. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages. J Innate Immun. 2013; 5: 591-602.31. Ramos-Kichik V, Mondragon-Flores R, Mondragon-Castelan M, et al. Neutrophil extracellular traps are induced by Mycobacterium tuberculosis. Tuberculosis (Edinb). 2009; 89: 29-37.32. Gamberale R, Giordano M, Trevani AS, Andonegui G and Geffner JR. Modulation of human neutrophil apoptosis by immune complexes. J Immunol. 1998; 161: 3666-74.33. Lu LL, Chung AW, Rosebrock TR, et al. A Functional Role for Antibodies in Tuberculosis. Cell. 2016; 167: 433-4334. Lu LL, Suscovich TJ, Fortune SM and Alter G. Beyond binding: antibody effector functions in infectious diseases. Nat Rev Immunol. 2017; 18: 46-61.35. Farga V and Caminero JA. Tuberculosis. . Mediterraneo 3ra ed. 2011.36. Kaufmann SH. How can immunology contribute to the control of tuberculosis? Nat Rev Immunol. 2001; 1: 20-30.37. Bozzano F, Marras F and De Maria A. Immunology of tuberculosis. Mediterr J Hematol Infect Dis. 2014; 6: e2014027.38. Corleis B, Korbel D, Wilson R, Bylund J, Chee R and Schaible UE. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils. Cell Microbiol. 2012; 14: 1109-21.39. Hilda NJ and Das S. Neutrophil CD64, TLR2 and TLR4 expression increases but phagocytic potential decreases during tuberculosis. Tuberculosis (Edinb). 2018; 111: 135-42.40. Aleyd E, van Hout MW, Ganzevles SH, et al. IgA enhances NETosis and release of neutrophil extracellular traps by polymorphonuclear cells via Fcalpha receptor I. J Immunol. 2014; 192: 2374-83.41. Mendez-Samperio P. Commentary: The Role of Neutrophils in the Induction of Specific Th1 and Th17 during Vaccination against Tuberculosis. Front Cell Infect Microbiol. 2017; 7: 179.42. Barberis I, Bragazzi NL, Galluzzo L and Martini M. The history of tuberculosis: from the first historical records to the isolation of Koch's bacillus. J Prev Med Hyg. 2017; 58: E9-E12.43. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ and Berry MP. The immune response in tuberculosis. Annu Rev Immunol. 2013; 31: 475-527.44. Kumar K and Kon OM. Diagnosis and treatment of tuberculosis: latest developments and future priorities. Annals of Research Hospitals. 2017; 1.45. Sua LF, Fernández, L. Tuberculosis miliar o diseminada. Revista Colombiana de Neumología. 2015; 27: 208 - 10.46. Kleinnijenhuis J, Oosting M, Joosten LA, Netea MG and Van Crevel R. Innate immune recognition of Mycobacterium tuberculosis. Clin Dev Immunol. 2011; 2011: 405310.47. Forrellad MA, Klepp LI, Gioffre A, et al. Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013; 4: 3-66.48. INS. Instituto Nacional de Salud. Tuberculosis: Protocolos de vigilancia y salud publica 2016.49. INS. Instituto Nacional de Salud. Guía para la vigilancia por laboratorio de tuberculosis. 2017.50. OPS. Organización Panamericana de Salud. MANUAL para el diagnóstico bacteriológico de la tuberculosis 2008.51. Pai M, Denkinger CM, Kik SV, et al. Gamma interferon release assays for detection of Mycobacterium tuberculosis infection. Clin Microbiol Rev. 2014; 27: 3-20.52. Garcia R, Lado FL, Bastida V, Pérez ML and Ortiz A. Tratamiento actual de la tuberculosis. An Med Interna. 2003; 20: 91-100.53. Li W, Deng G, Li M, Liu X and Wang Y. Roles of Mucosal Immunity against Mycobacterium tuberculosis Infection. Tuberc Res Treat. 2012; 2012: 791728.54. Liu CH, Liu H and Ge B. Innate immunity in tuberculosis: host defense vs pathogen evasion. Cell Mol Immunol. 2017; 14: 963-75.55. Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol. 2012; 12: 352-66. 56. Scordo JM, Knoell DL and Torrelles JB. Alveolar Epithelial Cells in Mycobacterium tuberculosis Infection: Active Players or Innocent Bystanders? J Innate Immun. 2016; 8: 3-14.57. Lerner TR, Borel S and Gutierrez MG. The innate immune response in human tuberculosis. Cell Microbiol. 2015; 17: 1277-85.58. McClean CM and Tobin DM. Macrophage form, function, and phenotype in mycobacterial infection: lessons from tuberculosis and other diseases. Pathog Dis. 2016; 74.59. Mihret A. The role of dendritic cells in Mycobacterium tuberculosis infection. Virulence. 2012; 3: 654-9.60. Allen M, Bailey C, Cahatol I, et al. Mechanisms of Control of Mycobacterium tuberculosis by NK Cells: Role of Glutathione. Front Immunol. 2015; 6: 508.61. Eruslanov EB, Lyadova IV, Kondratieva TK, et al. Neutrophil Responses to Mycobacterium tuberculosis Infection in Genetically Susceptible and Resistant Mice. Infection and Immunity. 2005; 73: 1744-53.62. Cummings BS and Schnellmann RG. Measurement of cell death in mammalian cells. Curr Protoc Pharmacol. 2004; Chapter 12: Unit 12 8.63. Driss EK, János, G.F. Modulation of neutrophil apoptosis and the resolution of inflammation through β2 integrins. Front Immunol. 2013; 4: 1-15.64. Tan BH, Meinken C, Bastian M, et al. Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens. J Immunol. 2006; 177: 1864-7165. Andersson H, Andersson B, Eklund D, et al. Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages. PLoS One. 2014; 9.66. Dheda K, Schwander SK, Zhu B, van Zyl-Smit RN and Zhang Y. The immunology of tuberculosis: from bench to bedside. Respirology. 2010; 15: 433-50.67. Lin PL and Flynn JL. CD8 T cells and Mycobacterium tuberculosis infection. Semin Immunopathol. 2015; 37: 239-49.68. Kozakiewicz L, Phuah J, Flynn J and Chan J. The role of B cells and humoral immunity in Mycobacterium tuberculosis infection. Adv Exp Med Biol. 2013; 783: 225-50.69. Abebe F and Bjune G. The protective role of antibody responses during Mycobacterium tuberculosis infection. Clin Exp Immunol. 2009; 157: 235-43.70. Rao M, Valentini D, Poiret T, et al. B in TB: B Cells as Mediators of Clinically Relevant Immune Responses in Tuberculosis. Clin Infect Dis. 2015; 61Suppl 3: S225-34.71. Ocampo M, Patarroyo MA, Vanegas M, Alba MP and Patarroyo ME. Functional, biochemical and 3D studies of Mycobacterium tuberculosis protein peptides for an effective anti-tuberculosis vaccine. Crit Rev Microbiol. 2014; 40: 117-45.72. The enzyme-linked immunosorbent assay (ELISA). Bulletin of the World Health Organization. 1976; 54: 129-39.73. Merrifield R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a tetrapeptide Journal of the American Chemical Society. 1963; 85: 2149-54.74. Houghten RA. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA. 1985; 82: 5131-5.75. Amersham Biosciences Limited. CNBr-activated Sepharose 4B. 2002, p. 1-12.76. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by co77. Andersson AM, Larsson M, Stendahl O and Blomgran R. Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner. J Innate Immun. 2019: 1-13.78. Larsen MH, Biermann, K, Jacobs, W.R. Laboratory Maintenance of Mycobacterium tuberculosis. Current Protocols in Microbiology. 2017; 6: 10A. 1. 1-A. 8.1.79. Perskvist N, Long M, Stendahl O and Zheng L. Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway. J Immunol. 2002; 168: 6358-65.80. Eum SY, Kong JH, Hong MS, et al. Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB. Chest. 2010; 137: 12281. Blomgran R, Desvignes L, Briken V and Ernst JD. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells. Cell Host Microbe. 2012; 11: 81-90.82. Kroon EE, Coussens AK, Kinnear C, et al. Neutrophils: Innate Effectors of TB Resistance? Front Immunol. 2018; 9: 2637.83. Lowe DM, Redford PS, Wilkinson RJ, O'Garra A and Martineau AR. Neutrophils in tuberculosis: friend or foe? Trends Immunol. 2012; 33: 14-25.84. Velmurugan K, Chen B, Miller JL, et al. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog. 2007; 3: e110.85. Lu LL, Smith MT, Yu KKQ, et al. IFN-gamma-independent immune markers of Mycobacterium tuberculosis exposure. Nat Med. 2019; 25: 977-87.86. Li H, Wang, X.X, Wang B, et al. Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis. Proc Natl Acad Sci 2017: 5023--887. Lu LL, Smith, M.T., Yu KKQ3, Luedemann C2, Suscovich TJ2, Grace PS2, Cain A2, Yu WH2,4, McKitrick TR5, Lauffenburger D4, Cummings RD5, Mayanja-Kizza H6, Hawn TR3, Boom WH7, Stein CM7,8, Fortune SM1,2, Seshadri C9, Alter G10. IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure Nat Med. 2019; 25: 977-87.88. Zimmermann N, Thormann V, Hu B, et al. Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis. EMBO Mol Med. 2016; 8: 1325-39.Derechos Reservados -Universidad Colegio Myor de Cundinamarca ,2019https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2Apoptosis de neutrófilos mediada por anticuerpos dirigidos contra péptidos derivados de proteínas de mycobacterium tuberculosis h37rvTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionColombiaEnfermedades micobacterianasEpidemiologíaTuberculosis pulmonarTuberculosisMycobacterium tuberculosisNeutrófilosAnticuerposApoptosis.ORIGINALAPOPTOSIS DE NEUTRÓFILOS MEDIADA POR ANTICUERPOS DIRIGIDOS CONTRA PÉPTIDOS DERIVADOS DE PROTEÍNA.pdfAPOPTOSIS DE NEUTRÓFILOS MEDIADA POR ANTICUERPOS DIRIGIDOS CONTRA PÉPTIDOS DERIVADOS DE 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accessunicolmayor/307oai:repositorio.unicolmayor.edu.co:unicolmayor/3072021-07-01 03:00:14.501An error occurred on the license name.|||https://creativecommons.org/licenses/by-nc-sa/4.0/open accessBiblioteca Digital Unicolmayorrepositorio@unicolmayor.edu.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