RESUMO
Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B(AU)
Assuntos
Oligonucleotídeos , Leucemia Linfocítica Crônica de Células BRESUMO
Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B
Assuntos
Leucemia Linfocítica Crônica de Células B , OligonucleotídeosRESUMO
We have investigated methods for modulating immune responses, against herpes simplex virus (HSV), generated from DNA vaccination by co-delivery of genes encoding costimulatory molecules. A strong delayed-type hypersensitivity (DTH) reaction was induced in mice co-injected via the intradermal (i.d.) route with a eukaryotic expression plasmid encoding the CD80 molecule (pCD80) and a plasmid encoding the glycoprotein D of the HSV-2 (pgD). Furthermore, when spleen cells from these mice were cultured in the presence of inactivated HSV, a significant increase in the expression of interleukin-2 receptor (IL-2R) was observed in the CD4 subset compared with mice immunized only with pgD. Analysis of cytokine synthesis at the single-cell level indicated that CD80 genes induce a significant increase in the number of interferon-gamma (IFN-gamma)-, IL-2- and IL-4-secreting cells in the spleen. On the other hand, co-administration of the CD80 gene via the intramuscular (i.m.) route did not induce an increase in the cell-mediated immune response. When a plasmid carrying the CD86 gene (pCD86) was co-injected via the i.m. route with the pgD plasmid, a small decrease in the number of IFN-gamma-secreting cells was observed. This down-regulation of the immune response was also observed when eukaryotic expression cassettes for CD80 and for CD86 were co-administered with the pgD plasmid via the i.d. route. However, co-injection of pCD86 via the i.m. route produced a small increase in the number of IL-4-secreting cells. When immunized mice were challenged intravaginally with 100 plaque-forming units of virus, only co-injection of the CD80 gene by the i.d. route provoked an adjuvant effect compared with mice immunized with pgD alone. A reduction in the titres of HSV in vaginal washings was observed together with a decrease in the lesion score.
Assuntos
Adjuvantes Imunológicos , Herpesvirus Humano 2/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Técnicas de Cultura de Células , Feminino , Herpes Genital/prevenção & controle , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Imunização , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral/imunologiaRESUMO
In the present report we established antigen dosages that induce oral tolerance of Th1 and Th2 lymphocytes or instead prime B- and Th2-dependent immune response and induce the tolerance of Th1 lymphocytes. Using different hapten-carrier systems, we found that low doses of OVA-DNP administered orally primed B and Th2 cells. On the other hand, no priming of B or Th2 cells was found in high-dose-OVA-DNP-fed rats. Low-dose-OVA-DNP-fed rats showed a strong mucosal immune response, with a high number of IgA anti-DNP antibody-forming cells in the lamina propria, while no mucosal immune response was observed in high-dose-OVA-DNP-fed rats. Thirty days after the immunization, tolerization of Th1 lymphocytes was confirmed in low- and high-dose-OVA-DNP-fed rats by diminished antigen-specific proliferation in vitro, reduced titers of anti-DNP IgG2a in serum, reduced expression of CD25 and CD134 molecules in cultured cells exposed to the antigen, reduced DTH reaction, and reduced IL-2 synthesis in culture. On the other hand, a high dose of OVA-DNP led to Th1 and Th2 tolerance, with an inhibition of specific IgG1 and IgG2a anti-DNP antibodies in serum after a parenteral challenge with OVA in CFA. This functional evidence was supported by the direct examination of IL-2 and IL-4 production. Furthermore, whereas in vitro assays seem to indicate that active suppression could be the responsible for Th1 tolerization in low-dose-OVA-DNP-fed rats, the results obtained after the transference of spleen or MLN cells to naive recipients support the idea that a subtractive mechanism is behind the tolerization of Th1 lymphocytes.
Assuntos
Antígenos/administração & dosagem , Linfócitos B/imunologia , Dinitrofenóis/administração & dosagem , Dinitrofenóis/imunologia , Tolerância Imunológica , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Células Th2/imunologia , Administração Oral , Transferência Adotiva , Animais , Formação de Anticorpos , Citocinas/biossíntese , Relação Dose-Resposta Imunológica , Feminino , Imunidade nas Mucosas , Técnicas In Vitro , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária , Ratos , Ratos Wistar , Baço/citologia , Baço/imunologia , Células Th1/imunologiaRESUMO
To study the importance of the bone marrow in the long-term antibody response, IgG and IgA antitoxin antibody-forming cells were evaluated by ELISPOT in Peyer's patches, mesenteric lymph nodes, spleen, lamina propria of the small intestine and bone marrow at several times after oral immunization with cholera toxin. The mesenteric lymph node was the site having the major frequency of IgG antitoxin during the first two weeks after priming, whereas lamina propria was the site with a major number of IgA antitoxin antibody-forming cells. However, from 3 weeks until 10 months after priming, bone marrow became the site with the major frequency of IgG, and especially IgA antitoxin antibody-forming cells (without taking into account the lamina propria). This result indicates that bone marrow was responsible for the long-term antibody response and raises questions concerning the mechanisms involved in the maintenance of antibody production. The importance of bone marrow as a site of antibody production was great when we analysed results as the true contribution of the total number of antitoxin antibody-forming cells, taking into account the number of cells recovered from each organ. When we analysed the anatomical location of memory B and T cells by adoptive transference, we found that cells from mesenteric lymph nodes and spleen were able to transfer a strong antibody response to naive syngeneic recipients, whereas bone marrow cells transferred a weak antibody response.
Assuntos
Antitoxinas/biossíntese , Linfócitos B/imunologia , Medula Óssea/imunologia , Toxina da Cólera/imunologia , Memória Imunológica , Administração Oral , Animais , Feminino , Imunidade nas Mucosas , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Intestino Delgado/imunologia , Linfonodos/imunologia , Nódulos Linfáticos Agregados/imunologia , Ratos , Baço/imunologia , Linfócitos T/imunologia , VacinaçãoRESUMO
In the present report the results indicate that the oral administration of one dose of CT in rats results in an antibody immune response in the spleen 48 h later, whereas no antitoxin antibody forming cells were found in the Peyer patches (PP), mesenteric lymph node (MLN) and lamina propria (LP) of the small intestine. At this time the main isotype of the antitoxin antibodies in the spleen were IgG and IgM, 5 days after the priming, few antitoxin AFC were observed in the MLN, IgG being the main isotype, whereas no IgM antitoxin AFC were found. At 1 week after priming the number of antitoxin AFC in the MLN reached similar values to those observed in the spleen. When cells from the spleen of rats primed orally with one dose of CT were cultured during 4 days in the presence of inhibitory doses of anti-Ia MAb (OX6), the number of antitoxin AFC was diminished when compared with that observed when cells were cultured in the absence of anti-Ia. The main isotype of antitoxin AFC observed when cells were analyzed after culture was IgM and it was the isotype most affected by the treatment with MAb anti-Ia. These results strongly suggest that an in situ presentation of the antigen did occur in the spleen. On the other hand, when the secondary immune response was studied 48 h after boosting, antitoxin AFC were found in the PP, MLN, SP and LP and 5 days after the booster a 20-30-fold increase was observed in all lymphoid tissues studied, indicating that the secondary immune response found in the spleen was mainly due to the recruitment of memory cells from Peyer's patches. However, when spleen cells were cultured 48 h after the immunization in the presence of inhibitory doses of anti-Ia a little decrease in the number of AFC was observed when compared with the controls (in absence of anti-Ia). The analysis of the antitoxin antibodies in sera and intestinal fluids were in line with the results presented above. The results shown in this report indicate that the systemic immune response observed after the oral administration of CT could be due in part to an in situ presentation of the antigen in the systemic compartments, especially in the spleen.
Assuntos
Anticorpos Antibacterianos/biossíntese , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Baço/imunologia , Administração Oral , Animais , Células Apresentadoras de Antígenos/imunologia , Antitoxinas/imunologia , Imunidade nas Mucosas , Cinética , Ratos , Ratos Wistar , Baço/efeitos dos fármacos , Fatores de Tempo , Vibrio choleraeRESUMO
The purpose of this study was to investigate in rats, by double-label immunofluorescence and flow cytometric analysis, the age related changes in the CD4 subset of gut-associated lymphoid tissues and spleen. We found that the percentage of CD4+ T cells in Peyer's patches (PP) and spleen (SP) increased during the first 6 weeks after weaning. An age-related decrease of the CD4 subset was observed in SP of aged rats, but not in their PP. In all lymphoid tissues studied, an age-related decrease of the Thy-1+ subset was observed from weaning to 2 years of age. Analysis of the naive CD4 subset (CD45RC+) showed that in SP this subset increased during the first 9 weeks of age, and declined in aged rats. However, in PP this subset presented a slow decrease from weaning until 2 years of age. Together with the decrease of the naive subset, a sharp increase of the memory/activated CD4+ cells (CD45RC- Thy-1-) was observed in PP, and to a lesser extent in SP. When the maturation of the CD4 T cells in PP was followed during the first week after weaning, we found that an important proportion of this subset changes its phenotype at this time, from recent thymic emigrant (CD45RC- Thy-1+) to naive T cell (CD45RC+ Thy-1-) and then to activated/memory cell (CD45RC- Thy-1-). Therefore it appeared that CD4 T cells from PP mature faster than SP CD4 T cells, and they are not subject to the deleterious effect of aging. One surprising point was the different kinetics of the CD4 T cells observed in mesenteric lymph nodes (MLN). No age-related changes were observed in the CD4 subset at this site. Furthermore, the percentage of the CD45RC+ cells did not decrease in aged rats, and in the first 9 weeks of life an increase of this subset was observed.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/citologia , Tecido Linfoide/citologia , Ratos , Ratos Wistar , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/citologia , DesmameRESUMO
Attempts to achieve IgA responses in the intestine by oral immunization with non replicating antigens have been characterized by ineffective responses of short duration unless long term dosages are administered. Cholera toxin (CT) is an exception in that it is able to produce a high secretory and systemic immune response. We study the effects of a bacterial immunomodulator [3 x 10(10) Propionibacterium granulosum ml-1 and lipopolysaccharide (LPS) 5 mg ml-1] on the immune response to CT orally administered to Wistar rats. The immunomodulator was orally administered as follows: in schedule 1 during 7 days prior to the first dose of CT; and in schedule 2, 2 days before, together, and 3 days after the first dose of CT. Schedules 1 and 2 were effective in increasing the specific IgA in the intestinal fluid and specific IgG in serum (P < 0.001) when compared to controls. Besides, schedule 2 was more effective than schedule 1 when the levels of specific IgG in serum or specific IgA in intestinal fluid was measured (P < 0.05). Total IgA in the intestinal fluid was increased in rats receiving the immunomodulator (P < 0.01). However, the ratio of specific IgA per total IgA was higher in rats receiving treatment 1 or 2 when compared to controls (P < 0.01). The number of antitoxin antibody producing cells was not increased in the Peyer patches, but a significant increase was observed in the mesenteric lymph nodes and spleen when compared to controls (P < 0.05). The administration of LPS alone produced an increase in the antitoxin immune response when compared to controls, but it was lower than those produced by the administration of the immunomodulator. These results indicate that this immunomodulator is an effective adjuvant of the mucosal and systemic immune response to CT. The mechanisms of action possibly involve nonespecific and specific modulations of the immune response.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Toxina da Cólera/administração & dosagem , Propionibacterium/imunologia , Administração Oral , Animais , Anticorpos/análise , Anticorpos/sangue , Líquido da Lavagem Broncoalveolar/imunologia , Toxina da Cólera/imunologia , Intestinos/imunologia , Ratos , Ratos Wistar , Baço/imunologiaRESUMO
To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry. After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01). These alterations remained even after 3 weeks of refeeding with stock diet. The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur. At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus. When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery. When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001). These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids. When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low. When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values. These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.
Assuntos
Animais Lactentes/imunologia , Linfócitos B/imunologia , Toxina da Cólera/imunologia , Mucosa Intestinal/imunologia , Distúrbios Nutricionais/imunologia , Nódulos Linfáticos Agregados/imunologia , Linfócitos T/imunologia , Animais , Antitoxinas/biossíntese , Antitoxinas/sangue , Antitoxinas/química , Linfócitos B/metabolismo , Peso Corporal/imunologia , Imunização , Secreções Intestinais/imunologia , Linfonodos/citologia , Mesentério , Nódulos Linfáticos Agregados/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
To study the importance of the bone marrow in the production of specific antibodies after a mucosal immunization with cholera toxin, the IgG, IgA and IgM specific antibody forming cells were evaluated by ELISPOT in Peyer patches, mesenteric lymph node (MLN), spleen, blood and bone marrow (BM). When 50-day-old rats were immunized intra-Peyer patches, a similar number of IgG and IgA antitoxin antibody forming cells (AFC) were found in the BM, whereas in the other lymphoid tissues a higher number of IgG antitoxin AFC were found. In all sites the peak of AFC was obtained 2 weeks after immunization. The administration of CT to 35-week-old rats resulted in a stronger immune response in all lymphoid tissues studied, but the proportion of antitoxin AFC contributed by the BM had not changed. One oral dose of cholera toxin resulted in a low number of antitoxin AFC, whereas when two or three doses of CT were administered orally an increase in the number of AFC was observed in the BM, reaching similar or higher numbers of IgG and IgA AFC than in the spleen. In all cases the highest number of AFC/10(6) cells was observed in the MLN, whereas antitoxin AFC were not found in the blood. The total number of AFC recovered from each organ was calculated taken into account that the BM of one femur represents 9% of the total BM. So, it was found that the BM is an important site in the production of IgG antitoxin antibodies, being the main site in the IgA antitoxin antibody production.