Modulación de la activación de células dendríticas por antígenos de Fasciola hepatica y ligandos para receptores Toll: Potencial terapéutico en respuestas anti-inflamatorias. Fasciola hepatica antigens and TLR ligands modulate dendritic cells activation: therapeutic potential in anti-inflammatory responses.

Antecedentes: En nuestro laboratorio hemos demostrado que antígenos (Ags) de Fasciola hepatica inducen en células dendríticas murinas (CD), diferentes propiedades tolerogénicas como la incapacidad por si mismos de inducir la maduración de las células, la resistencia a la maduración por ligandos de TLR, el incremento en la producción de IDO y también la capacidad de esta estas células de dirigir la respuesta inmune hacia un perfil Th2 y T reg. Por otra parte ha sido bien documentado que CD con características tolerogénicas, ya sea inmaduras o semimaduras, son útiles para reducir respuestas inflamatorias excesivas tales como las que ocurren en enfermedades autoinmunes. Además hemos demostrado que CD tratadas con Ags del parásito en conjunto con un ligando Toll (CpG-ODN) producen altos niveles de citoquinas anti-inflamatorias (IL-10 y TGF-) bajos de citoquinas proinflamatorias (TNF, IL-6, IL-12). Hipótesis: El fenotipo semimaduro alcanzado en las CDpodría ser utilizado para reducir la inflamación en un modelo de enfermedad autoinmune en donde existe una exacerbada respuesta Th1 y Th17, ya que la producción elevada de IL-10 y TGF- podría inhibir o controlar estas respuestas de manera directa o a través de la inducción de células T regulatorias. Objetivos: En este proyecto nosotros proponemos la inmunización de animales susceptibles (ratones DBA1/j), al desarrollo de artritis inducida por colágeno (AIC) con CD tratadas con Ags de F. hepatica en conjunto con CpG-ODN para reducir los síntomas clínicos de la enfermedad. Materiales a utilizar: En nuestro laboratorio hemos desarrollado un modelo de artritis inducida por colágeno (AIC) mediante dos inmunizaciones de ratones DBA1/j con colágeno tipo II bovino y adyuvante de Freund. El modelo permitió establecer un índice clínico mediante la hinchazón en las patas de los animales. Doce días posteriores a la primera inmunización los animales serán inyectados con CD tratadas con: 1. PBS, 2.Extracto total de F.hepatica (TE) + CII, 3. CpG + CII, 4. TE+CpG+CII Se realizará la observación macroscópica diaria, a partir de los 7 días de la 2a inmunización Luego del sacrificio las articulaciones de las patas se prepararán para realizar un análisis histológico. Se detectará en suero los niveles de anticuerpos IgG1 (perfil Th2) y de IgG2a (perfil Th1) mediante la técnica de ELISA. Se detectará también el perfil de citoquinas en los nódulos drenantes por la técnica de ELISA y adicionalmente la poblaciónes celulares de células T regulatorias (Treg) CD4+CD25+Foxp3 o células Tr1. Resultados esperados: Pensamos que el tratamiento de los animales que desarrollan AIC con CD semimaduras (por el tratamiento con TE y CpG), serán capaces de migrar a los órganos linfaticos y secretar TGF-be(inductora de células T reg), IL-10 (inductoras de células Tr1), IDO inhibitoria de la respuesta de Li T y promotor de células T reg, también podría generarse una respuesta Th2 (por la presencia de antígenos del parásito), y estas respuestas aisladas o en forma sinérgica podrían inhibir las respuestas de tipo Th17 y Th1 asociadas a la patología en esta enfermedad. Importancia del proyecto: En el desarrollo de la artritis existe un aumento de la inmunidad mediada por células, asi como de la respuesta inmune humoral hacia componentes de la matriz del cartílago. El tratamiento convencional de la artritis recae en general en el uso de inmunosupresores no-específicos, los cuales poseen una variedad de efectos adversos y la inhibición de la respuesta inflamatoria no es específica. En este proyecto proponemos el uso de CD tratadas con antígenos del helminto F. hepatica y CpG ligando Tol que capacita a estas células para generar una respuesta adaptativa de tipo regulatoria, útil en la inhibición de las respuestas inflamatorias como la que ocurre durante la progresión de artritis reumatoidea en un modelo experimental en ratones. We have shown that F. hepatica Ags-treated dendritic cells (DC) together with a TLRl ligand (CpG-ODN) produce high levels of anti-inflammatory cytokines (IL-10 and TGF-Beta) and low of proinflammatory cytokines (TNF, IL-6, IL -12). Hypothesis: The semimature phenotype achieved by DC, could be used to reduce inflammation in a model of autoimmune disease. The high production of IL-10 and TGF-Beta by these cells could directly or through the induction of T reg cells inhibit the inflammatory response. Objective: In this project we propose the immunization of DBA1 / j mice, susceptible to the development of collagen-induced arthritis (CIA) with F. hepatica-treated DC in conjunction with CpG-ODN to reduce clinical signs of disease. Materials: In our laboratory, we developed the CIA model by two immunizations of DBA1 / j mice with bovine type II collagen and Freund's adjuvant. The model allowed to stablish a clinical index by swelling in the legs of animals. Twelve days after the first immunization the animals are injected with DC treated with: 1. PBS 2. F.hepatica Extract (TE) + CII, 3. CpG + CII, 4. TE + CpG + CII Macroscopic observation will take place daily from 7 days of the 2nd immunization. After sacrifice the joints of the legs will be prepared for histological analysis. Serum levels of IgG1 antibodies (Th2 profile) and IgG2a (Th1 profile) will be detected by ELISA. It will also detected the cytokine profile in draining lymph nodes by ELISA and additionally the cell populations of regulatory T cells (Treg) CD4 + CD25 + Foxp3 or Tr1 cells. Expected results: We believe that the treatment of animals that had developed CIA with DC will be able to migrate to lymphatic organs and secrete TGF-B (T reg cell-inducing), IL-10 (inducing Tr1 cells), IDO (inhibitory of T cells and inducing of T reg cells) could alone or in synergy inhibit Th17-type responses and Th1 associated with the pathology in this disease.

Trypanosoma cruzi: circulating antigens

Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.

Development of cell mediated immunity to flagellar antigens and acquired resistance to infection by Trypanosoma cruzi in mice

Modulation by BCG and/or cyclophosphamide of sensitization of mice with flagellar fraction (a tubulin-enriched fraction) prevented death of mice challenged with T. cruzi CL strain trypomastigotes recovered from Vero cells. A methodology was ceveloped to assay specific antigens and to determine optimal doses for sensitization and elicitation of DTH in mice. CL strain is predominantly myotropic strain which does not produce important parasitism of mononuclear phagocyte cells; these cells appear to control infection when activated in vivo. Maximum protection was seen in this study when BCG and cyclophosphamide were associated, but protection was observed also when cyclophosphamide, that prevents supressor T cells, was applied 2 days before flagellar fraction sensitization in normal mice. These experiments suggested that the macrophage may have an important role in the early phases of infection particularly when nonspecific stimulation is associated with specific sensitization. A correlation betwen delayed hypersensitivity to parasite antigens and protection was observed.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

The use of monoclonal antibodies for the characterization and production of Mycobacterium leprae antigens

Similar immunizations of mice and hybridoma technology were used by several investigators to raise monoclonal antibodies which identified a limited range of epitopes and antigenic molecules. Further studies would have the scope for revealing yet more novel structures. The existing MABs are agreed standard reagents, avaiable to investigators and valuable for several applications. At least six epitopes specific for M. leprae were defined in molecular terms. Monoclonal antibody based immunoassays proved to be invaluable for the screening of recombinant DNA clones and for the topographic study of individual epitopes. Purification of antigens using affinity chromatography requires further development of techniques whilst serology of leprosy is open for clinical and epidemiological evaluation.

T-cell response to my mycobacterial antigens in lepromatous and tuberculoid leprosy

We showed that a large fraction of lepromatous patients do harbor helper-type circulating T-cells that can be activated in vitro by Mycobacterium leprae. M. leprae and PPD triggered T-cell lines could be then obtained from both tuberculoid and lepromatous patients. The proliferative response of these helper T-cells is predominantly directed against epitopes shared by several species of mycobacteria, in lepromatous patients as well as in tuberculoid patients, but species specific T-cells are also present. When presented in the context of M. leprae, these cross reactive epitopes usually fail to stimulate the T-cell lines of lepromatous patients, because of the contamination of the lines by supressor T-cells actavable by M. leprae. In one lepromatous patient, PPD and M. leprae reactive T-cell lines and clones (of the CD4 phenotype), exhibited a strong cytotoxic activity to autologous target cells coated with antigen: the relevance of this phenomenon to the pathophysiology of lepromatous leprosy remains however unknown.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human Schistosomiasis: I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.