Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-gamma levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-beta was high, whereas IL-12 and TNF-alpha were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.

Ocular toxoplasmosis: the role of retinal pigment epithelium migration in infection.

Our aim was to study the migration of retinal pigmented epithelium (RPE) into the retinal layer during infection of C57BL/6 mice with Toxoplasma gondii. Eyes from infected and non-infected animals were analyzed on the 60th day of infection by light and transmission electron microscopy. Non-infected eyes showed a normal morphology. In contrast, we observed free parasites in the retinal vasculature, the presence of mononuclear inflammatory infiltrate (MNII) and parasites in the vasculature of choroids in infected eyes. No inflammatory infiltrate was observed; RPE cells were identified near the MNII in nuclear and plexiforme layers. RPE cells were also found on the ganglion cell layer and in the outer segments of the photoreceptor. The morphology showed that RPE cells caused a discontinuity in the nuclear and plexiforme layers. Clusters of parasites were found surrounded by RPE cells and MNII in the inner plexiforme layers. Ultrastructural analysis showed that RPE cells migrated through the epithelium into the inner retinal layers. We did not observe Toxoplasma cysts in many eyes in which pathological changes were detected. Only 8.3% of the animals had Toxoplasma cysts in the inner nuclear layer in the absence of inflammatory cells. The migration of RPE cells can be triggered by a disruption of the RPE monolayer or injury to the neural retina, as in the case of toxoplasmosis.

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection.

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.

Study of acute chagasic mice under immunosuppressive therapy by cyclosporin A : modulation and confocal analysis of inflammatory reaction.

The in vivo effects of cyclosporin A (CsA) on Trypanosoma cruzi infection were examined using different schedules of the drug in mice infected with the Y strain. Parasitaemia at day 8 after infection among CsA-treated animals was usually higher than control infected non-treated mice. On the other hand, mortality analysis showed that animals CsA-treated either with 200 mg/kg 2 days before infection or with therapeutic doses (10 mg/kg every other day) showed almost the same mean time of death (35.8 and 38.2 days, respectively). In these groups mice died 50% less than control infected non-treated ones. The mean time of death in the animals treated with 200 mg/kg 5 days after infection and in infected non-treated control mice were respectively 29.0 and 22.6 days. The kinetics analysis of the leukocyte population of animals treated with a single dose of 200 mg/kg of CsA before or after infection did not show the alternate pattern of leukopenia/leukocytosis observed in control groups of infected mice but differential cell counts indicated a modulatory action upon circulating leukocytes of therapeutic doses of CsA. The animals treated with any of the CsA schedules showed a moderate to intense diffuse inflammatory reaction exhibiting mainly mononuclear cells in the heart. Immunofluorescence analysis by confocal microscopy revealed that macrophages are a major component of the inflammatory infiltrate in all groups of CsA-treated mice and also in the control group.