Omega-3 Polyunsaturated Fatty Acids Prevent Toxoplasma gondii Infection by Inducing Autophagy via AMPK Activation.

Omega-3 polyunsaturated fatty acids (ω3-PUFAs) have potential protective activity in a variety of infectious diseases, but their actions and underlying mechanisms in Toxoplasma gondii infection remain poorly understood. Here, we report that docosahexaenoic acid (DHA) robustly induced autophagy in murine bone marrow-derived macrophages (BMDMs). Treatment of T. gondii-infected macrophages with DHA resulted in colocalization of Toxoplasma parasitophorous vacuoles with autophagosomes and reduced intracellular survival of T. gondii. The autophagic and anti-Toxoplasma effects induced by DHA were mediated by AMP-activated protein kinase (AMPK) signaling. Importantly, BMDMs isolated from Fat-1 transgenic mice, a well-known animal model capable of synthesizing ω3-PUFAs from ω6-PUFAs, showed increased activation of autophagy and AMPK, leading to reduced intracellular survival of T. gondii when compared with wild-type BMDMs. Moreover, Fat-1 transgenic mice exhibited lower cyst burden in the brain following infection with the avirulent strain ME49 than wild-type mice. Collectively, our results revealed mechanisms by which endogenous ω3-PUFAs and DHA control T. gondii infection and suggest that ω3-PUFAs might serve as therapeutic candidate to prevent toxoplasmosis and infection with other intracellular protozoan parasites.

Increased expressions of ADAMTS-13 and apoptosis contribute to neuropathology during Toxoplasma gondii encephalitis in mice.

Toxoplasma gondii (T. gondii) is a protozoan parasite with the potential of causing severe encephalitis among immunocompromised humans and animals. Our previous study showed that T. gondii induces high nitric oxide (NO) production, high glial activation (GFAP) and neurofilament expressions, leading to severe neurodegeneration in toxoplasma encephalitis (TE) in the central nervous system (CNS). The aim of this experimental study was to investigate ADAMTS-13 expression and apoptosis in CNS and to identify whether they have any correlation with toxoplasmosis neuropathology and neurodegeneration. Mice were infected with ME49 strain T. gondii and the levels of ADAMTS-13, caspase 3, caspase 8, caspase 9, TNFR1 and Bcl-xL expressions were examined in brain tissues by immunohistochemistry, during the development and establishment of chronic infections at 10, 30 and 60 days post-infection. Results of the study revealed that the levels of ADAMTS-13 (P < 0.005), caspase 3 (P < 0.05), caspase 8 (P < 0.05), caspase 9 (P < 0.005) and TNFR1 (P < 0.05) expressions in the brain markedly increased while Bcl-xL expression decreased (P < 0.005). The most prominent finding from our study was that 10, 30 and 60 days post-infection ADAMTS-13 increased significantly and this may play an important role in the regulation and protection of the blood-brain barrier integrity and CNS microenvironment in TE. These results also suggest that T. gondii-mediated apoptosis might play a pivotal role and a different type of role in the mechanism of neurodegeneration and neuropathology in the process of TE. Furthermore, expression of ADAMTS-13 might give an idea of the progress and is critical for diagnosis of this disease. To the best of the authors' knowledge, this is the first report on ADAMTS-13 expression in the CNS of T. gondii-infected mice.

Role of IDO activation in anti-microbial defense in human native astrocytes.

The most serious complication of human toxoplasmosis is the development of toxoplasmic encephalitis. It is well established that in the brain Toxoplasma gondii is able to replicate in microglial cells, astrocytes and neurons, and that all three cell types can harbor toxoplasma cysts. The role of astrocytes in the defense against toxoplasma is not clear. The most prominent effector-mechanisms against toxoplasma are the induction of the inducible form of the nitric oxide synthase (iNOS), and the induction of indoleamine 2,3-dioxygenase (IDO). In this paper we show that interferon (IFN)-gamma-activated, native human astrocytes express IDO activity, as shown by the detection of IDO mRNA using RT-PCR, detection of enzyme expression with IDO-specific monoclonal antibodies in Western blots, as well as by direct measurement of enzyme activity in the activated cells. IFN-gamma-mediated IDO activity in human astrocytes inhibits the growth of Toxoplasma gondii and of group B streptococci. Furthermore, we show for the first time that IFN-gamma induced IDO activity is also effective in inhibiting the growth of Herpes Simplex Virus in astrocyte cultures. In addition, iNOS expression was detectable by RT-PCR in all batches of astrocytes tested when stimulated with a cytokine cocktail of IFN-gamma, TNF-alpha, IL-1 and LPS. Furthermore, we found that the amount of nitric oxide produced by astrocytes is not sufficient to inhibit either toxoplasmal or bacterial growth. Co-activation of iNOS and IDO on the other hand, results in an inhibition of IDO activity in astrocytes.

Kinetics of the nucleoside triphosphate hydrolase of Toxoplasma gondii in mice with acute and chronic toxoplasmosis.

The kinetics of the nucleoside triphosphate hydrolase (NTPase) of Toxoplasma gondii was examined using an avidin-biotin sandwich-ELISA (ABS-ELISA) based on an anti-NTPase monoclonal antibody, 6C6. The RH and ME49 strains of the parasite were used to produce acute and chronic infections in mice, respectively. In the acute model, detectable serum concentrations of NTPase were observed from day 1 post-infection and gradually increased until the death of the mice. They were associated with parasitaemia (as estimated by bioassay). No anti-T. gondii antibody could be detected at any time. In the chronic model, in which 20 T. gondii ME49 cysts were given to each mouse per os, the NTPase concentration generally increased from day 3, peaked between days 7 and 14 and then declined. However, one of the four mice used still had a high serum concentration of NTPase on day 35. Again, detectable NTPase concentrations occurred when the mice had parasitaemias. Antibody to T. gondii was detected from day 7 (IgM) or 10 (IgG) and brain cysts were observed from day 14. Since detectable serum concentrations of NTPase appear to be associated with parasitaemia in both acute and chronic toxoplasmosis, the ABS-ELISA of the enzyme may make a useful diagnostic tool.

Increased activity of matrix metalloproteinases in the cerebrospinal fluid of patients with HIV-associated neurological diseases.

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.

Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice.

Infection of C57BL/6 mice with Toxoplasma gondii leads to progressive and ultimately fatal chronic Toxoplasma encephalitis (TE). Genetic deletion or inhibition of inducible nitric oxide synthase (iNOS) from the beginning of infection increased the number of T. gondii cysts in the brain and markedly reduced the time-to-death in this mouse strain. In the present study, we addressed whether iNOS also contributes to the control of intracerebral parasites in a clinically stable latent infection that develops in T. gondii-resistant BALB/c mice after resolution of the acute phase of TE. iNOS was expressed in the inflammatory cerebral infiltrates of latently infected BALB/c mice, but the number of iNOS+ cells was significantly lower than in the brains of chronically infected T. gondii-susceptible C57BL/6 mice. In BALB/c mice with latent TE (> 30 days of infection), treatment with the iNOS inhibitors L-N6-iminoethyl-lysine or L-nitroarginine-methylester for < or = 40 days did not result in an increase of the intracerebral parasitic load and a reactivation of the disease, despite the presence of iNOS-suppressive inhibitor levels in the brain. However, L-nitroarginine-methylester treatment had remarkably toxic effects and induced a severe wasting syndrome with high mortality. In contrast to BALB/c mice, L-N6-iminoethyl-lysine treatment rapidly exacerbated the already established chronic TE of C57BL/6 mice. Thus, the containment of latent toxoplasms in T. gondii-resistant BALB/c mice is independent of iNOS, whereas the temporary control of intracerebral parasites in T. gondii-susceptible C57BL/6 mice with chronic TE requires iNOS activity.