Joseli Lannes-Vieira

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-γ as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi

We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.

Modulation of Chemokine Production and Inflammatory Responses in Interferon-γ- and Tumor Necrosis Factor-R1-Deficient Mice during Trypanosoma cruzi Infection

Infection with Trypanosoma cruzi causes a strong inflammatory reaction at the inoculation site and, later, in the myocardium. The present study investigates the role of cytokines as modulators of T. cruzi-induced chemokine expression in vivo and in vitro. In macrophage cultures, although the stimulation with interferon (IFN)-gamma increases the expression of IP-10, it blocks KC expression. Tumor necrosis factor (TNF)-alpha, on the other hand, potentiates KC, IP-10, macrophage inflammatory protein-1alpha, and JE/monocyte chemotatic protein-1 expression. Interleukin-10 and transforming growth factor-beta inhibited almost all chemokines tested. The role of IFN-gamma and TNF-alpha in chemokine modulation during infection was investigated in T. cruzi-infected IFN-gamma-deficient (GKO) or TNF-R1/p55-deficient (p55-/-) mice. The expression of chemokines detected in the inoculation site correlated with the infiltrating cell type observed. Although GKO mice had a delayed and intense neutrophilic infiltrate correlating with the expression of KC and macrophage inflammatory protein-2, none of the above was observed in p55-/- mice. The detection of infiltrating T cells, Mig, and IP-10 in the myocardium was observed in wild-type and p55-/-, but not in GKO mice. Together, these results suggest that the regulatory roles of IFN-gamma and TNF-alpha on chemokine expression may play a crucial role in the modulation of the inflammatory response during T. cruzi infection and mediate resistance to infection.

Extracellular matrix proteins in intrathymic T-cell migration and differentiation?

Intrathymic T-cell migration and differentiation is not completely understood. Here, Wilson Savino and colleagues argue that certain interactions between differentiating thymocytes and thymic epithelial cells are mediated by extracellular matrix proteins and that these interactions influence intrathymic migration events and thymocyte differentiation.

Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) Antagonist (Met-RANTES) Controls the Early Phase of Trypanosoma cruzi–Elicited Myocarditis

Background—Comprehension of the pathogenesis of Trypanosoma cruzi–elicited myocarditis is crucial to delineate strategies aimed at ameliorating the inflammation associated with heart dysfunction. The augmented expression of CC chemokines, especially CCL5/RANTES and CCL3/MIP-1&agr;, in the hearts of infected mice suggests a role for CC chemokines and their receptors in the pathogenesis of T cruzi–elicited myocarditis. Methods and Results—We report that during the early phase of infection in C3H/HeJ mice infected with 100 blood trypomastigotes of T cruzi, most of the inflammatory cells invading the heart tissue were CD8+ cells and expressed CCR5, a CCL5/RANTES, and CCL3/MIP1-&agr; receptor. Furthermore, peripheral blood CD8+ T lymphocytes displayed increased expression of CCR5. These findings led us to use Met-RANTES, a selective CCR1 and CCR5 antagonist, to modulate the acute T cruzi–elicited myocarditis. Met-RANTES treatment did not interfere with parasitism but significantly decreased the numbers of CD4+ and CD8+ T cells, CCR5+, and interleukin-4+ cells invading the heart, paralleling the diminished deposition of fibronectin. Moreover, Met-RANTES treatment resulted in increased survival of infected animals, compared with saline treatment. Conclusions—These results indicate that the massive influx of CCR5+ cells into cardiac tissue is not crucial for cell-mediated anti–T cruzi immunity but appears to be critical for pathogenesis of T cruzi–elicited myocarditis. Thus, CC chemokine receptors might become an attractive therapeutic target for further evaluation during T cruzi infection.

Protective effect of a naked DNA vaccine cocktail against lethal toxoplasmosis in mice.

BALB/c mice were intramuscularly immunized with low doses (25-50microg) of DNA cocktail containing plasmids encoding the full-length SAG1/P30 and the 196-561 terminal sequence of ROP2 genes. This immunization resulted in a Th1-type response with predominance of IgG2a and a specific T-cell proliferation with high levels of interferon-gamma (IFN-gamma) secretion, whereas no IL-4 was detected. Moreover, DNA cocktail immunization induced a long-lasting protection against a lethal challenge with the highly virulent Toxoplasma gondii RH strain, whereas low doses of single genes were not protective. These results support further investigations to achieve a multigene anti-T. gondii DNA vaccine.

Extracellular matrix components of the mouse thymus microenvironment: ontogenetic studies and modulation by glucocorticoid hormones.:

The present investigation was an ontogenetic study on the distribution of extracellular matrix (ECM) components in the thymic microenvironment of C57BL/6 mice (comprising young and old adults and developing embryos) and NZB mice. In addition, we evaluated the in vivo and in vitro influence of hydrocortisone treatment on basement membrane protein production by a thymic epithelial cell line. In young normal animals, Type I collagen was restricted to the interstitial spaces of the capsule and septa, where Type IV collagen, fibronectin, and laminin could be detected in the basement membranes. In addition, fibronectin-containing fibers were seen within the medulla of the thymic lobules. The ECM distribution pattern in the developing embryos was distinct from that observed in adults, since a fine meshwork of basement membrane-containing proteins was clearly seen throughout the parenchyma. Moreover, aging normal and NZB mice exhibited a denser ECM pattern than young adult normal animals. Treatment with hydrocortisone, both in vivo and in vitro, resulted in enhancement of ECM expression, detected in mice as early as 2 hr post injection and lasting for several days. Considering that the fluctuations of ECM expression parallel important events in thymocyte differentiation, we discuss the possibility that the two phenomena may be associated.

Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination

ABSTRACT A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.

Soluble Factors Released by Toxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration

ABSTRACT The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2 (PGE2) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2 and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.

Oxidative stress fuels Trypanosoma cruzi infection in mice

Oxidative damage contributes to microbe elimination during macrophage respiratory burst. Nuclear factor, erythroid-derived 2, like 2 (NRF2) orchestrates antioxidant defenses, including the expression of heme-oxygenase-1 (HO-1). Unexpectedly, the activation of NRF2 and HO-1 reduces infection by a number of pathogens, although the mechanism responsible for this effect is largely unknown. We studied Trypanosoma cruzi infection in mice in which NRF2/HO-1 was induced with cobalt protoporphyrin (CoPP). CoPP reduced parasitemia and tissue parasitism, while an inhibitor of HO-1 activity increased T. cruzi parasitemia in blood. CoPP-induced effects did not depend on the adaptive immunity, nor were parasites directly targeted. We also found that CoPP reduced macrophage parasitism, which depended on NRF2 expression but not on classical mechanisms such as apoptosis of infected cells, induction of type I IFN, or NO. We found that exogenous expression of NRF2 or HO-1 also reduced macrophage parasitism. Several antioxidants, including NRF2 activators, reduced macrophage parasite burden, while pro-oxidants promoted it. Reducing the intracellular labile iron pool decreased parasitism, and antioxidants increased the expression of ferritin and ferroportin in infected macrophages. Ferrous sulfate reversed the CoPP-induced decrease in macrophage parasite burden and, given in vivo, reversed their protective effects. Our results indicate that oxidative stress contributes to parasite persistence in host tissues and open a new avenue for the development of anti-T. cruzi drugs.