UNIVERSIDAD DE BUENOS AIRES ...
Vaccination is the most effective method to control an infection. Effective vaccines induce a protective immune response by activating T and B lymphocytes, which, converted into memory cells, control infections caused by the pathogens included in the vaccine formulation. Vaccines made from inactivat...
Guardado en:
| Autor principal: | |
|---|---|
| Otros Autores: | |
| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2011
|
| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_1570 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_1570.dir/1570.PDF |
| Aporte de: |
| id |
I28-R145-HWA_1570 |
|---|---|
| record_format |
dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
| collection |
Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
| orig_language_str_mv |
spa |
| topic |
Bovinos Lipoarabinomananos Micobacterias Bovinos Respuesta inmune Lipoarabinomanano Micobacterias Inmunología |
| spellingShingle |
Bovinos Lipoarabinomananos Micobacterias Bovinos Respuesta inmune Lipoarabinomanano Micobacterias Inmunología Colavecchia, Silvia UNIVERSIDAD DE BUENOS AIRES ... |
| topic_facet |
Bovinos Lipoarabinomananos Micobacterias Bovinos Respuesta inmune Lipoarabinomanano Micobacterias Inmunología |
| description |
Vaccination is the most effective method to control an infection. Effective vaccines induce a protective immune response by activating T and B lymphocytes, which, converted into memory cells, control infections caused by the pathogens included in the vaccine formulation. Vaccines made from inactivated viruses or bacteria or subunits have the ability to generate an efficient specific immune response. However, this response is sometimes weak because the viruses or bacteria do not multiply inside the host. Because of this, the use of adjuvants is required to enhance the immune response. The selection of an appropriate adjuvant can not only enhance the immune response, but also modulate the immunoglobulin isotype and the type of profile (Th1 or Th2) produced. Lipoarabinomannan (LAM) is non-covalently bound to the plasma membrane of mycobacteria and extends to the outer part of the cell wall. This molecule plays an important role in the physiology of bacteria as well as in the modulation of the host immune response during infection. Several groups of researchers have described the effects of mycobacteria as adjuvants and modulators inducing a Th1 response when added to vaccines. The aim of this thesis was to evaluate the effect of the application of ovalbumin (OVA) together with LAM and incomplete Freund's adjuvant (IFA) on the specific cellular and humoral immune response of bovines to OVA. LAM was extracted and purified from Mycobacterium avium subsp. avium using a chemical extraction, as previously (Hamasur et al., 1999). The yield obtained was similar to that obtained by those authors. Using the monoclonal antibody that specifically recognizes LAM of Mycobacterium tuberculosis, we confirmed that the structure of LAM obtained from cultures of Mycobacterium avium subsp. avium retains a high degree of antigenic\nsimilarity to that of Mycobacterium tuberculosis.\nA total of 22 crossbred calves between 10 and 12 months of age were\nused. Calves were randomly assigned to four treatment groups: G-IFA,\nimmunized with PBS and 1 mL of IFA; G-LAM, immunized with LAM (1\nmg/dose) and IFA; G-OVA, immunized with OVA (1 mg/dose) and IFA; and\nG-OVALAM, immunized with OVA, IFA and LAM. Animals were immunized\nsubcutaneously on days 0, 21 and 42. Since one of the most important limiting factors for the use of adjuvants is the possible direct or indirect toxic\neffect on products derived from human consumption, a number of physiological parameters were evaluated to control the formulation of the immunogen used.\nThe analysis of the these parameters (hematological values, complete\nblood count, serum protein profile, weight gain, and reactivity to purified\nprotein derivative of Mycobacterium avium subsp. avium (PPA)) indicated that the treatment with LAM did not induce detectable alterations. This could be a first approach for the assessment of the safety of the immunomodulator. We then evaluated the innate immune response by\ndetecting the production of reactive oxygen intermediates in adherent\nperipheral cells at time 0 (pre-immunization) and final time (15 days postthird\nimmunization) and detected a decrease in the production of the reactive oxygen intermediates in the G-OVALAM group at the final time. The modulation produced in the cellular immune response was\nassessed by the proliferation of mononuclear cells stimulated with mitogens\nand antigens, lymphocyte subpopulations in peripheral blood, IFN production at time 0 and final time, and specific skin hypersensitivity. A significant increase was detected in the proliferative response of the animals belonging to the group G-OVALAM, indicating that LAM increases the specific\ncellular immune response with mitogens such as ConA. An increase in IFN? production was observed in culture supernatants of\nmononuclear cells stimulated in vitro with OVA and ConA, in animals treated\nwith LAM at levels of between 600 and 800 pg/mL. An increase in the\npercentage of cells with the CD25 marker on the surface was also observed.\nThe expression of this marker is associated with the increase in\nsubpopulations with regulatory functions, although these functions have not been demonstrated in cattle. Since the specific response against purified\nprotein derivative of Mycobacterium bovis could not be detected in groups\ninoculated with LAM as an immunomodulator, the results indicate that\nmodulation by LAM does not interfere with the diagnosis of tuberculosis in\nvivo or with that of paratuberculosis in vitro.\nThe kinetics of the specific antibody response against OVA was evaluated on days 0, 21, 42 and 57, whereas the response of isotypes was analyzed using bovine IgM, IgG1, IgG2 and IgG3 antibodies. The results\nshow that the modulation by LAM in cattle induced specific high titers of\nantibodies as from the first immunization. These levels increased with the\nsecond dose and remained at the same level after the third immunization.\nThe serum response against OVA was predominantly IgG1 and IgG2, and\nIgG3 to a lesser extent. The IgG1/IgG2 relationship showed a decrease in\nthe animals immunized with LAM. This suggests a trend of LAM to induce a\nTh1 response. This thesis allowed determining the effect of the addition of LAM to the incomplete Freund's adjuvant as a potential modulator to a Th1 response\nagainst OVA when it is simultaneously inoculated. |
| author2 |
Mundo, Silvia Leonor |
| author_facet |
Mundo, Silvia Leonor Colavecchia, Silvia |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Colavecchia, Silvia |
| author_sort |
Colavecchia, Silvia |
| title |
UNIVERSIDAD DE BUENOS AIRES ... |
| title_short |
UNIVERSIDAD DE BUENOS AIRES ... |
| title_full |
UNIVERSIDAD DE BUENOS AIRES ... |
| title_fullStr |
UNIVERSIDAD DE BUENOS AIRES ... |
| title_full_unstemmed |
UNIVERSIDAD DE BUENOS AIRES ... |
| title_sort |
universidad de buenos aires ... |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2011 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_1570 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_1570.dir/1570.PDF |
| work_keys_str_mv |
AT colavecchiasilvia universidaddebuenosaires AT colavecchiasilvia lipoarabinomananosdemicobacteriasefectossobrelarespuestainmuneenbovinos |
| _version_ |
1824356438257958912 |
| spelling |
I28-R145-HWA_15702024-08-27 UNIVERSIDAD DE BUENOS AIRES ... Vaccination is the most effective method to control an infection. Effective vaccines induce a protective immune response by activating T and B lymphocytes, which, converted into memory cells, control infections caused by the pathogens included in the vaccine formulation. Vaccines made from inactivated viruses or bacteria or subunits have the ability to generate an efficient specific immune response. However, this response is sometimes weak because the viruses or bacteria do not multiply inside the host. Because of this, the use of adjuvants is required to enhance the immune response. The selection of an appropriate adjuvant can not only enhance the immune response, but also modulate the immunoglobulin isotype and the type of profile (Th1 or Th2) produced. Lipoarabinomannan (LAM) is non-covalently bound to the plasma membrane of mycobacteria and extends to the outer part of the cell wall. This molecule plays an important role in the physiology of bacteria as well as in the modulation of the host immune response during infection. Several groups of researchers have described the effects of mycobacteria as adjuvants and modulators inducing a Th1 response when added to vaccines. The aim of this thesis was to evaluate the effect of the application of ovalbumin (OVA) together with LAM and incomplete Freund's adjuvant (IFA) on the specific cellular and humoral immune response of bovines to OVA. LAM was extracted and purified from Mycobacterium avium subsp. avium using a chemical extraction, as previously (Hamasur et al., 1999). The yield obtained was similar to that obtained by those authors. Using the monoclonal antibody that specifically recognizes LAM of Mycobacterium tuberculosis, we confirmed that the structure of LAM obtained from cultures of Mycobacterium avium subsp. avium retains a high degree of antigenic\nsimilarity to that of Mycobacterium tuberculosis.\nA total of 22 crossbred calves between 10 and 12 months of age were\nused. Calves were randomly assigned to four treatment groups: G-IFA,\nimmunized with PBS and 1 mL of IFA; G-LAM, immunized with LAM (1\nmg/dose) and IFA; G-OVA, immunized with OVA (1 mg/dose) and IFA; and\nG-OVALAM, immunized with OVA, IFA and LAM. Animals were immunized\nsubcutaneously on days 0, 21 and 42. Since one of the most important limiting factors for the use of adjuvants is the possible direct or indirect toxic\neffect on products derived from human consumption, a number of physiological parameters were evaluated to control the formulation of the immunogen used.\nThe analysis of the these parameters (hematological values, complete\nblood count, serum protein profile, weight gain, and reactivity to purified\nprotein derivative of Mycobacterium avium subsp. avium (PPA)) indicated that the treatment with LAM did not induce detectable alterations. This could be a first approach for the assessment of the safety of the immunomodulator. We then evaluated the innate immune response by\ndetecting the production of reactive oxygen intermediates in adherent\nperipheral cells at time 0 (pre-immunization) and final time (15 days postthird\nimmunization) and detected a decrease in the production of the reactive oxygen intermediates in the G-OVALAM group at the final time. The modulation produced in the cellular immune response was\nassessed by the proliferation of mononuclear cells stimulated with mitogens\nand antigens, lymphocyte subpopulations in peripheral blood, IFN production at time 0 and final time, and specific skin hypersensitivity. A significant increase was detected in the proliferative response of the animals belonging to the group G-OVALAM, indicating that LAM increases the specific\ncellular immune response with mitogens such as ConA. An increase in IFN? production was observed in culture supernatants of\nmononuclear cells stimulated in vitro with OVA and ConA, in animals treated\nwith LAM at levels of between 600 and 800 pg/mL. An increase in the\npercentage of cells with the CD25 marker on the surface was also observed.\nThe expression of this marker is associated with the increase in\nsubpopulations with regulatory functions, although these functions have not been demonstrated in cattle. Since the specific response against purified\nprotein derivative of Mycobacterium bovis could not be detected in groups\ninoculated with LAM as an immunomodulator, the results indicate that\nmodulation by LAM does not interfere with the diagnosis of tuberculosis in\nvivo or with that of paratuberculosis in vitro.\nThe kinetics of the specific antibody response against OVA was evaluated on days 0, 21, 42 and 57, whereas the response of isotypes was analyzed using bovine IgM, IgG1, IgG2 and IgG3 antibodies. The results\nshow that the modulation by LAM in cattle induced specific high titers of\nantibodies as from the first immunization. These levels increased with the\nsecond dose and remained at the same level after the third immunization.\nThe serum response against OVA was predominantly IgG1 and IgG2, and\nIgG3 to a lesser extent. The IgG1/IgG2 relationship showed a decrease in\nthe animals immunized with LAM. This suggests a trend of LAM to induce a\nTh1 response. This thesis allowed determining the effect of the addition of LAM to the incomplete Freund's adjuvant as a potential modulator to a Th1 response\nagainst OVA when it is simultaneously inoculated. Fil: Colavecchia, Silvia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires. Argentina Mundo, Silvia Leonor Colavecchia, Silvia 2011-03 .La vacunación es el método más efectivo para controlar una infección.Las vacunas efectivas inducen una respuesta inmune protectora, activando\nlinfocitos T y B, que convertidos en células de memoria controlan infecciones producidas por el patógeno incluido en la formulación de la vacuna. Las vacunas elaboradas con virus o bacterias inactivados o a subunidades tienen\nla capacidad de generar una respuesta inmune específica eficiente, pero a veces débil ya que no se multiplican en el huésped. Es por eso, que\nrequieren el uso de adyuvantes que potencien la respuesta inmune. La\nselección de un adyuvante apropiado no solamente permite aumentar la\nrespuesta inmune sino que también permite modular el isotipo de inmunoglobulina que se produce y también el tipo de perfil, Th1 o Th2.\nEl Lipoarabinomanano (LAM) se encuentra unido de forma no covalente\na la membrana plasmática de las micobacterias y se extiende hasta el\nexterior de la pared celular. Esta molécula juega un papel muy importante en\nla fisiología de las bacterias así como también en la modulación de la\nrespuesta inmune del huésped durante la infección. Numerosos grupos de\ninvestigadores describen los efectos adyuvantes y moduladores de las\nmicobacterias induciendo la respuesta hacia un perfil Th1 cuando se agregan\na vacunas.\nEl objetivo de este trabajo de tesis fue evaluar el efecto de la aplicación\nde ovalbúmina (OVA) junto con LAM y adyuvante de Freund incompleto (AFI)\nen la respuesta inmune celular y humoral de bovinos específica a OVA.\nSe extrajo y purificó LAM de Mycobacterium avium subsp. avium\nutilizando una extracción química publicada previamente por Hamasur et al. (1999), y se obtuvo un rendimiento similar al obtenido por dichos autoresMediante el uso del anticuerpo monoclonal que reconoce específicamente\nLAM de Mycobacterium tuberculosis se confirmó que la estructura del LAM\nobtenido a partir de cultivos de Mycobacterium avium subsp. avium conserva\nun alto grado de similitud antigénica con el de Mycobacterium tuberculosis.\nSe utilizaron 22 terneros mestizos de entre 10 y 12 meses de edad los\ncuales fueron asignados al azar a 4 grupos de tratamiento, G-AFI,\ninmunizados con PBS y 1 mL de AFI, G-LAM, inmunizados con LAM 1\nmg/dosis y AFI, G-OVA, inmunizados con OVA (1 mg/dosis) y AFI, y GOVALAM, inmunizados con OVA, AFI y LAM. Se inmunizaron en forma\nsubcutánea a los días 0, 21 y 42. Dado que una de las limitantes más importantes del uso de adyuvantes es el posible efecto tóxico directo o indirecto sobre los productos derivados al consumo humano se evaluaron una serie de parámetros fisiológicos a fin de controlar la formulación del inmunógeno utilizado. El análisis de los parámetros evaluados (valores\nhematológicos, fórmula leucocitaria absoluta, perfil de proteínas séricas,\nganancia de peso, reactividad a derivado proteico purificado de\nMycobacterium avium subsp. avium (PPA)) indicó que el tratamiento con\nLAM no indujo alteraciones detectables, esto podría constituir un primer\nacercamiento a la evaluación de la inocuidad del inmunomodulador. Se\nevaluó la respuesta inmune innata a través de la detección de la producción\nde intermediarios reactivos de oxígeno en células periféricas adherentes a\ntiempo 0 (preinmunización) y a tiempo final (15 días post-tercera inmunización) y se ha detectado una disminución de la producción de los intermediarios reactivos del oxígeno en el grupo G-OVALAM evaluado a tiempo final. La modulación producida en la respuesta inmune celular se evaluó a través de la proliferación de células mononucleares estimuladas con mitógenos y antígenos, las subpoblaciones de linfocitos en sangre periférica, la producción de IFN? a tiempo 0 y tiempo final y la hipersensibilidad cutánea. específica. Se detectó un incremento en la respuesta proliferativa de aquellos animales pertenecientes al grupo G-OVALAM indicando que el LAM incrementa la respuesta inmune celular específica y con mitógenos como ConA. Se ha detectado un aumento en la producción de IFN? en\nsobrenadantes de cultivo de células mononucleares estimuladas in vitro con\nOVA y ConA, en los animales tratados con LAM con niveles de entre 600 y\n800 pg/mL. Se obtuvo además, un incremento en el porcentaje de células\ncon el marcador CD25 en superficie. La expresión de este marcador se\nrelaciona con el incremento de subpoblaciones con funciones reguladoras\naunque aún estas funciones no se han demostrado en bovinos. Dado que la\nrespuesta específica frente a derivado proteico purificado de Mycobacterium\nbovis no pudo ser detectada en los grupos inoculados con LAM como\ninmunomodulador, los resultados obtenidos indican que la modulación por\nLAM no interfiere con el diagnóstico de tuberculosis in vivo ni en el de\nparatuberculosis in vitro. Se evaluó la cinética de respuesta específica de\nanticuerpos frente a OVA a los días 0, 21, 42 y 57 y la respuesta de isotipos\nutilizando anticuerpos anti IgM, IgG1, IgG2 e IgG3 bovinos. Los resultados\nobtenidos muestran que la modulación por LAM en bovinos indujo\nanticuerpos específicos de alto título desde la primera inmunización. Estos\nniveles se incrementaron en la segunda dosis y se mantuvieron luego de la\ntercera inmunización. La respuesta sérica predominante frente a OVA es IgG1 e IgG2 y en menor proporción IgG3. La relación entre IgG1/IgG2\nmostró una disminución en los animales inmunizados junto con LAM. Esto\nsugiere una tendencia de LAM a inducir una respuesta de tipo Th1. Este trabajo de tesis nos permitió conocer el efecto del agregado de\nLAM al adyuvante de Freund incompleto como posible modulador hacia una\nrespuesta de tipo Th1 frente a OVA inoculada simultáneamente. application/pdf Bovinos Lipoarabinomananos Micobacterias spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Bovinos Respuesta inmune Lipoarabinomanano Micobacterias Inmunología Inmunología Doctora de la Universidad de Buenos Aires en Ciencias Veterinarias Lipoarabinomananos de micobacterias : efectos sobre la respuesta inmune en bovinos info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_1570 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_1570.dir/1570.PDF |