RESUMEN
Vaccination against pathogens involved in bovine respiratory disease (BRD) is a useful tool to reduce the risk of this disease however, it has been observed that the commercially available vaccines only partially prevent the infections caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, it is recommended to search for new adjuvant strategies to minimise the economic impact of this respiratory syndrome. A possibility to improve the conventional vaccine response is to modulate the immune system with probiotics, since there is accumulating evidence that certain immunomodulatory strains administered around the time of vaccination can potentiate the immune response. Considering veterinary vaccines are frequently tested in murine models, we have developed an immunisation schedule in BALB/c mice that allows us to study the immune response elicited by BRD vaccine. In order to evaluate a potential strategy to enhance vaccine efficacy, the adjuvant effect of Enterococcus faecalis CECT7121 on the murine specific humoral immune response elicited by a commercial vaccine against BRD was studied. Results indicate that the intragastric administration of E. faecalis CECT7121 was able to induce an increase in the specific antibody titres against the bacterial components of the BRD vaccines (P. multocida and M. haemolytica). The quality of the humoral immune response, in terms of antibody avidity, was also improved. Regarding the cellular immune response, although the BRD vaccination induced a low specific secretion of cytokines in the spleen cell culture supernatants, E. faecalis CECT7121-treated mice showed higher interferon-γ production than immunised control mice. Our results allowed us to conclude that the administration of E. faecalis CECT7121 could be employed as an adjuvant strategy to potentiate humoral immune responses.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas Bacterianas/inmunología , Complejo Respiratorio Bovino/prevención & control , Enterococcus faecalis , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/inmunología , Probióticos , Animales , Anticuerpos Antibacterianos/sangre , Afinidad de Anticuerpos/inmunología , Vacunas Bacterianas/administración & dosificación , Complejo Respiratorio Bovino/inmunología , Bovinos , Citocinas/biosíntesis , Femenino , Esquemas de Inmunización , Ratones , Ratones Endogámicos BALB C , Pasteurellaceae/inmunología , Probióticos/administración & dosificaciónRESUMEN
AIMS: To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response. METHODS AND RESULTS: Enterococcus faecalis CECT7121 showed a high adhesion capacity to completely and heterogeneously differentiated human intestinal epithelial cell line (Caco-2 cells). In addition, the contact of this bacterium with Caco-2 cells did not induce inflammatory chemokines (IL-8 and CCL-20). The presence of IgA(+) and IL-6(+) cells in the small intestine, as well as the production of inflammatory cytokines (TNFα, IL-6 and IL-12) in the gut, was determined after intragastric inoculation of Ent. faecalis CECT7121 in BALB/c mice. The administration of Ent. faecalis CECT7121 increased the number of IgA(+) cells in the intestinal lamina propria without modifying the percentage of IL-6(+) cells. No differences were observed in the cytokines measured in the intestinal extracts between probiotic-treated and control mice. CONCLUSIONS: Enterococcus faecalis CECT7121 stimulates local mucosal immunity and adheres to IECs without inducing inflammatory signals. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that, apart from its already reported systemic immune activity, Ent. faecalis CECT7121 has a modulatory effect at a local level.
Asunto(s)
Enterococcus faecalis/fisiología , Células Epiteliales/inmunología , Inmunidad Mucosa/efectos de los fármacos , Mucosa Intestinal/inmunología , Intestinos/inmunología , Probióticos/administración & dosificación , Animales , Células CACO-2 , Citocinas/biosíntesis , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Humanos , Interleucina-6/inmunología , Interleucina-8/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Intestinos/efectos de los fármacos , Intestinos/microbiología , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
INTRODUCTION: Previous studies showed that placental extracts (PE) alleviates arthritic symptoms in animal models of arthritis. METHODS: To evaluate whether murine PEs obtained at embryonic days 7.5 (PE7) and 17.5 (PE18) regulate RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured with RANKL and MCSF in presence or not of PEs. Tartrate-resistant acid phosphatase (TRAP) was stained and multinucleated TRAP positive cells were visualized under a light microscope. Cathepsin K and metalloprotease expression was assessed by RT-PCR and gelatin zymography respectively. NFATc1 expression was determined by immunoblot. To analyze NFAT-dependent transcription, macrophages were transfected with a luciferase reporter plasmid. Cytokines were determined in PEs by ELISA and immunoblot. Transforming growth factor (TGF)- beta and Interleukin (IL)-10 receptor were inhibited in cell cultures with specific antibodies. RESULTS: PE7 and PE18 inhibited RANKL-induced multinucleated TRAP positive cells, Cathepsin K expression and metalloprotease activity, as well as NFATc1 expression and activity, thereby inhibiting osteoclast differentiation of RAW cells. Inflammatory/Regulatory cytokine ratio was higher in PE7 than in PE18. Blocking TGF-beta abolished the effect of both, PE7 and PE18, on multinucleated TRAP positive cells and metalloprotease expression, whereas blocking IL-10 receptor reverted the effect of PE18 but not of PE7. DISCUSSION: Inhibition of osteoclast differentiation by PEs was not unexpected, since cytokines detected in extracts were previously found to regulate osteoclast differentiation. CONCLUSIONS: PEs inhibited osteoclast differentiation of macrophages in vitro. Downregulation of NFATc1 might be involved in this effect. Regulatory/Th2 cytokines play a role in the effect of PEs on osteoclast differentiation.
Asunto(s)
Citocinas/farmacología , Macrófagos/citología , Osteoclastos/citología , Placenta/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Catepsina K/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/análisis , Embrión de Mamíferos , Femenino , Isoenzimas/metabolismo , Macrófagos/efectos de los fármacos , Metaloproteasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Placenta/química , Embarazo , Ligando RANK/farmacología , Fosfatasa Ácida Tartratorresistente , Extractos de Tejidos/análisis , Extractos de Tejidos/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
The increased production of asymmetric IgG protective antibodies is one of the mechanisms proposed to explain a successful semiallogeneic pregnancy. We have previously demonstrated that IL-6 was able to enhance the synthesis of these antibodies by a murine hybridoma, while the glucocorticoid dexamethasone (DEXA) diminished it. In order to investigate the mechanism of asymmetric antibody synthesis, we investigate the role of UDP-Glc glycoprotein glucosyltransferase (GT), an endoplasmic reticulum enzyme involved in the quality control and folding of glycoproteins. Either recombinant murine rmIL-6 (0-10-40-160-320-640 ng/ml) or DEXA (0.15 microM) were added to a mouse hybridoma culture and incubated for 24 and 72 h in the first case, and for 4h in the presence of DEXA. Anti-DNP asymmetric antibodies were determined in the culture supernatants by ELISA. After harvesting, hybridoma cells were sonicated and GT activity was analysed in isolated microsomal fractions by measuring UDP((14)C)-Glc incorporation into urea-denatured thyroglobulin (urea-Tg). In the present paper, we showed that IL-6, mainly at 40 ng/ml and t=24h, was able to upregulate both in vitro GT activity (+74%) and asymmetric molecule synthesis (+227%). Lower increases were obtained employing 10 and 160 ng/ml. On the other hand, DEXA, at 0.15 microM and t=4h, showed a mild inhibition of enzyme activity (-10%) and a diminished proportion of asymmetric IgG (-49%). A direct relationship between GT activity and proportion of the asymmetric antibody synthesised was found in our hybridoma cells employing IL-6 and DEXA in different conditions, as was indicated by a correlation analysis. These results suggest that GT might be involved in the synthesis of asymmetric antibodies.
Asunto(s)
Dexametasona/farmacología , Glucosiltransferasas/metabolismo , Inmunoglobulina G/biosíntesis , Interleucina-6/farmacología , Animales , Hibridomas , Ratones , Regulación hacia Arriba , Uridina Difosfato/metabolismoRESUMEN
PROBLEM: CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. METHOD OF STUDY: Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. RESULTS: CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. CONCLUSION: This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.
Asunto(s)
Aborto Espontáneo/tratamiento farmacológico , Heparina de Bajo-Peso-Molecular/uso terapéutico , Interleucina-6/biosíntesis , Interleucina-6/uso terapéutico , Placenta/metabolismo , Aborto Espontáneo/metabolismo , Animales , Técnicas de Cultivo , Femenino , Reabsorción del Feto/tratamiento farmacológico , Ratones , Placenta/citología , EmbarazoRESUMEN
PROBLEM: Protecting antibodies against trophoblast surface molecules were previously described. Here we analysed the synthesis of asymmetric IgG by placental B-lymphocytes. METHOD OF STUDY: B cells were isolated from human term placenta and cord blood, stimulated with anti-CD40 IgG and cocultured with transfected Fcgamma R-expressing mice Ltk-fibroblast. Interleukin-4, IL-6, IL-10, IL-11 and IL-13 were added to cultures for 14 days. Asymmetric IgG were assessed in culture supernatants by concanavalin A (Con A) fixation and enzyme-linked immunosorbent assay. RESULTS: When IL-6 was added to the cultures, the percentages of asymmetric IgG synthesized by placental B cells were: IL-6: 29 +/- 10; IL-6 + IL-10: 24 +/- 7; IL-4 + IL-10 + IL-6: 38 +/- 9. The last combination induced the highest increase in the asymmetric IgG synthesis as compared with control (19 +/- 10%, P < 0.05). Additionally, placental B cells synthesized more asymmetric IgG than umbilical cord blood B-lymphocytes (P = 0.0015). CONCLUSIONS: Isolated placental B-lymphocytes synthesized asymmetric IgG in response to Th2 interleukins, more notably IL-6 in combination with IL-4 and IL-10. The in vitro increase of protective asymmetric IgG synthesis in response to Th2-cytokines support the hypothesis that a local Th2-switch is beneficial for pregnancy outcome.
Asunto(s)
Formación de Anticuerpos/fisiología , Linfocitos B/inmunología , Interleucinas/metabolismo , Placenta/inmunología , Animales , Femenino , Sangre Fetal/inmunología , Humanos , Inmunoglobulina G/biosíntesis , RatonesRESUMEN
Rat spleen and peripheral blood lymphocytes express progesterone receptors whose concentration is increased greatly during the early phase of pregnancy. After stimulation of progesterone the expression of receptors was augmented 2-3 times. When cells were cultured in the presence of progesterone they released a soluble factor that inhibited cellular immunoreactions (MLR, CRC) and cellular proliferation as measured by thymidine incorporation by spleen-cell culture. This factor also inhibited the synthesis of anti-DNP antibodies by a mouse hybridoma and diminished the proportion of cells in phase S. However, the percentage of asymmetric molecules produced by the hybridoma remained unaltered. These results support the hypothesis that soluble factors released by rat lymphocytes modulate the immune response of the mother and participate in the mechanism that protects the fetus against antipaternal antibodies.
Asunto(s)
Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Intercambio Materno-Fetal/efectos de los fármacos , Intercambio Materno-Fetal/inmunología , Progesterona/farmacología , Animales , Formación de Anticuerpos , Medios de Cultivo Condicionados , Citosol/metabolismo , Citotoxicidad Inmunológica , Femenino , Humanos , Técnicas In Vitro , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Receptores de Progesterona/inmunología , Receptores de Progesterona/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
PROBLEM: To evaluate the effect of rat placental culture supernatants (PS) on spontaneous, mitogen- and alloantigen-induced lymphoproliferation, antibody synthesis regulation, and symmetric/asymmetric antibody ratio. METHOD OF STUDY: The effect of PS was determined: (a) on cell proliferation of murine hybridoma cells and on spontaneous or ConA-induced proliferation of murine and rat splenocytes by thymidine incorporation; (b) on rat or mouse cell-mediated cytotoxicity (CMC) by 51Cr release; and (c) on antibody synthesis by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: With 20% PS, hybridoma cell inhibition was 37% and that of splenocytes up to 60%, whereas it was 75 and 43%, respectively, in the presence of ConA. Despite marked cell death, hybridoma proliferation index increased significantly. There was a drop in total antidinitrophenylated (DNP) immunoglobulin G1 (IgG1) antibody production and an increase in asymmetric antibody percentage, correlating with placental supernatant concentration. CONCLUSIONS: Rat placental culture supernatants inhibit cell proliferation in all cases, diminish total antibody production, and increase the percentage of asymmetric antibodies by the hybridoma, and they increase antibody production by rat splenocytes.
Asunto(s)
Formación de Anticuerpos , Inmunidad Celular , Placenta/inmunología , Animales , Técnicas de Cultivo , Citotoxicidad Inmunológica , Femenino , Hibridomas/citología , Hibridomas/inmunología , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Bazo/citología , Bazo/inmunologíaRESUMEN
PROBLEM: To investigate how the factors secreted by human placenta modify the quality and the quantity of the antibody produced by the hybridoma as well as its cellular proliferation. METHOD: Supernatants of cultures of human placenta (PS) were added to a mouse IgG1 hybridoma culture producing anti-DNP antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. RESULT: It was found that PS augmented by 40-50% the quantity of total antibody produced, increased the proportion of asymmetric (blocking) antibodies from 15% to 30%, and diminished the cellular proliferation, as measured by 3H-thymidine incorporation. CONCLUSION: These results, together with other similar observations already described in human, rat and mouse pregnancies, suggest that secretory factors produced by the placenta do modify the immune response of the mother against paternal antigens and participate in the mechanisms that make possible the survival of the allogenic fetus.