By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1−/−/common cytokine receptor γ-chain (γCR)), and T and B cell and IFN-γ-deficient (RAG-1−/−/IFN-γ−/−) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-γ secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-γ-dependent innate immune protection. Inoculation of wild type, but not IFN-γ−/− bone marrow-derived macrophages protected RAG-1−/−/IFN-γ−/− mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1−/− C. pneumoniae-infected mice expressed IFN-γ mRNA. Reconstitution of RAG-1−/−/γcR−/− or RAG-1−/−/IFN-γ−/− mice with CD4+ or CD8+ cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-γ−/− CD4+ or IFN-γ−/− CD8+ SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-γR−/− CD4+ or IFN-γR−/− CD8+ SC were inoculated into RAG-1−/−/IFN-γ−/− mice. Thus both nonlymphoid and T cell-derived IFN-γ can play a central and complementary role in protection against C. pneumoniae. IFN-γ secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-γ regulated T cell protective functions.
The leaves of guaco (Mikania glomerata and M. laevigata) are widely used for the treatment of asthma and bronchitis. An LC method for the quantification of coumarin and O-coumaric acid in medicinal extracts was developed and validated for linearity, limit of detection, accuracy, precision, as well as intra- and inter-day variations. Extracts and isolated markers were tested in the mice allergic pneumonitis model and the histopathological profile of the lung tissue was analysed. The values found for coumarin and O-coumaric acid in a fluid extract were 1.53 and 1.69 mg/mL, respectively, for M. glomerata, and 0.96 and 0.38 mg/mL for M. laevigata. The values found for the lyophilised aqueous extract were 0.22 and 0.11 mg/mL of coumarin and O-coumaric acid in M. glomerata and 0.05 and 0.02 mg/mL in M. laevigata, respectively . The analysed samples from the species M. glomerata presented more coumarin and O-coumaric acid than the analogous M. laevigata species. Both coumarin and O-coumaric acid are part of the phytocomplex which is responsible for the therapeutic activity of the guaco species. The lyophilisation process generated some alterations in the extract, in comparison with the fresh aqueous extract, and these extracts did not present anti-inflammatory activity. Comparing the histopathological images of the groups tested, a haemorrhagic profile of lung tissue of animals treated with lyophilised extract, O-coumaric acid and coumarin is observed, but not for the group treated with hydroalcoholic extract. It is probable that some protective effect of the whole extract (lost during the lyophilisation process) blocks the harmful effects of the isolated markers.
The microbicidal activity of macrophages in an inflammatory milieu has been related to the production of a large number of cytokins and intermediary metabolites of oxygen and nitrogen among them, nitric oxide (NO). Considering that granulomatous inflammation is predominantly composed of macrophages and epithelioid cells, we decided to investigate the participation of NO in this peculiar type of inflammation. Two models were used: glass cover slip implantation into the subcutaneous tissue of mice and, the inoculation of live bacillus Calmette-Guérin (BCG) into the footpad of the animals. Using a histochemical method for the detection of NO synthase and of the concentration of citrulin metabolized by cells obtained from cover slips implanted on different time intervals or BCG-activated peritoneal cells, it was possible to demonstrate that epithelioid cells do not produce NO. Cells from granuloma induced by BCG inoculation express NO synthase, with different degrees of reactivity with a higher intensity in the cytoplasm of cells located in the edge of the lesions. The expression of NO synthase in the cytoplasm of these cells decreases with the age of the lesions. It could also be demonstrated that in mice treated with l-name, an inhibitor of NO metabolism, the lesions induced by BCG lost the granulomatous architecture, were necrotic, and had a significant increase in the bacillary load of the lesion. These data allow us to conclude that NO production by macrophages is a determining factor in the organization of the granulomatous lesion and that it also controls the bacterial load in BCG-induced lesions in mice.
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