Efecto in vitro e in vivo de un inmunomodulador compuesto por LPS de E. coli y Propionibacterium granulosum sobre el sistema inmune del cerdo
Immunomodulation is defined as the modification of the immune response by means of the administration of substances that possess the ability to regulate the immune system. INMD is an immunomodulator composed by a combination of a detoxified LPS of E. coli and inactivated cells of Propionibacterium granulosum that has demonstrated to have a modulatory effect on the immune system of the pig. In the present study we have done several in vitro and in vivo assays with the objective of evaluate the effects that this compound produces in some components of the immune system in pigs. In vitro assays were made to determine the action of INMD and their components on the cytokine production or expression in alveolar macrophages and peripheral blood mononuclear cells, the activation of phagocytosis in polymorphonuclear neutrophiles, the activation of lymphocytes through the study of the IL- 2 receptor (CD25) and the expression of the major histocompatibility complex class-II (MHC-II) in alveolar macrophages. Regarding the cytokine expression, it could be observed that alveolar macrophages expressed IL-1, IL-6, IL-12 and produced TNF-a, but not IL-10, while the peripheral blood mononuclear cells expressed IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and produced INF-g in response to INMD stimulation. When the effect of INMD on the phagocytosis exerted by PMN was studied, we observed that the phagocytosis capability of these cells increased. This phenomenon was attributable more to an increase in the number of activated cells than to an increase of individual phagocytic ability. This effect was correlated with P. granulosum. Likewise, we observed that INMD and its separated components induced the expression of MHC-II in alveolar macrophages. Thus as well in cells infected with Aujeszkys disease virus as in those that were not. Regarding CD25, we observed that INMD and its components by themselves alone were able to induce the expression of this molecule, a fact indicates the activation of the peripheral blood mononuclear cells. In this case, the maximum expression of CD25 was observed with INMD both in Aujeszkys disease virus infected cells as well as in uninfected ones. Two in vivo experiments were made. The first one of them the effect of the administration of INMD was evaluated using this product as a coadjuvant to the vaccination against the Aujeszkys disease virus. With this purpose, eighty-five 8-10-weeks-old crossbred conventional pigs were randomly distributed in two groups (group A = 43 and group B = 42), that were vaccinated two times against the Aujeszkys disease virus. Group B animals received simultaneously with the first vaccine dose, an intramuscular dose of INMD equal to 20 mg/ml of LPS and 250 mg/ml of P. granulosum. Blood was collected from all 85 animals before the first vaccination, before the booster and five weeks after the booster. Sera were analysed to determine the presence of Aujeszkys disease virus anti-gE and anti-gB antibodies, total immunoglobulin load (IgG1, IgG2) and Aujeszkys disease virus specific immunoglobulins (IgA, IgG1, IgG2). Lymphoproliferation assays and virus seroneutralization test for Aujeszkys disease virus were also done. Before the first vaccination, 97.6% of pigs in group A and 100% in group B presented anti-gB antibodies, while 11.6% and 19% were seropositive against gE respectively. At the end of the trial, 96.4 % of group A pigs and 87.1% of the animal in group B were gB+ and none of them had anti-gE antibodies. Regarding levels of total immnunoglobulins, after the first vaccination group B pigs had higher levels of IgG1 and IgG2 (p<0,01) but these differences disappeared after the second vaccination. When evaluated Aujeszkys disease virus specific antibodies, were not different between groups, except for IgA. In this case, we observed that after the revaccination, 38.48% of animals in group B presented titres £1:40, while in group A this was observed in the 14.81% of the pigs (p<0.01). In lymphoproliferation assays, significant differences between groups were not observed. Regarding the virus-neutralizing antibodies, we observed higher titres in the group B animals after de first vaccination (p<0.005), differences that disappeared after the booster. Thus after the first vaccination, 56.7% of pigs in group A had titres £1:4, while thus was observed only in 25.6% of the animals in group B (p<0.05). In a second in vivo assay, the adverse effects that could cause the application of INMD were evaluated. With this aim, eight pigs were distributed in three groups. Animals of group A were injected intramuscularly with a dose of INMD corresponding to 1.2 mg/kg of body weight of LPS and 16 mg/ml of body weight of P. granulosum, while those in group B received 2.6 mg/kg of body weight of LPS and 33 mg/kg of body weight of P. granulosum. Animals in group C received similar volume of sterile saline solution. All the animals were bled to determine the levels of TNF-a to the 0, 1, 2, 4, 6 and 24 hours, taking in each one of these times the body temperature. None of the animals had evident adverse effects for LPS or produced by high levels of cytokine. From those results it could conclude that INMD exerts its action on the immune system at least indicating cytokine expression, enhancing phagocytosis capabilities of PMNS and enhancing also the expression on MHC-II and CD25. These effects can contribute substantially improvement of the immune response of pigs in disease state or in vaccination.
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School:Universitat Autónoma de Barcelona
Source Type:Master's Thesis
Keywords:457 departament de salut i anatomia animals
Date of Publication:09/25/2002