BackgroundPreviously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8+ T cell responses in HLA-A0201+ individuals chronically infected by T. cruzi.Methods and FindingsHerein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8+ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8+ T cell responses to promiscuous epitopes revealed that the CD8+ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection.ConclusionsThis study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.

Previously we found that the frequency of IFN-γ-producing CD8+ T cells specific for Trypanosoma cruzi inversely correlates with disease severity in chronic human Chagas disease along with low levels of IL-2-secreting CD8+ T cells in all clinical stages. This impairment of the parasite-specific T cell responses was associated with phenotypic features of immune senescence of the CD8+ T cell compartment. These data prompted us to address the question of whether the CD4+ T cell compartment also experiences signs of exhaustion. Thus, we performed a functional and phenotypical characterization of T. cruzi-specific and overall CD4+ T cells in chronically infected subjects with different degrees of cardiac dysfunction. The results show an inverse association between disease severity and the frequency of T. cruzi-specific IFN-γ-producing CD4+ T cells. The high expression of CD27 and CD28 with a relative low expression of CD57 found on CD4+IFN-γ + T cells suggests that the effector T cell pool in chronic T. cruzi infection includes a high proportion of newly recruited T cells, but a low frequency of long-term memory cells. The total CD4+ T cell compartment shows signs of senescence and later stages of differentiation associated with more severe stages of the disease. These findings support the hypothesis that long-term T. cruzi infection in humans might exhaust long-lived memory T cells.

Novel methods for treatment of African trypanosomiasis, caused by infection with Trypanosoma brucei are needed. Cordycepin (3'-deoxyadenosine, 1a) is a powerful trypanocidal compound in vitro but is ineffective in vivo because of rapid metabolic degradation by adenosine deaminase (ADA). We elucidated the structural moieties of cordycepin required for trypanocidal activity and designed analogues that retained trypanotoxicity while gaining resistance to ADA-mediated metabolism. 2-Fluorocordycepin (2-fluoro-3'-deoxyadenosine, 1b) was identified as a selective, potent, and ADA-resistant trypanocidal compound that cured T. brucei infection in mice. Compound 1b is transported through the high affinity TbAT1/P2 adenosine transporter and is a substrate of T. b. brucei adenosine kinase. 1b has good preclinical properties suitable for an oral drug, albeit a relatively short plasma half-life. We present a rapid and efficient synthesis of 2-halogenated cordycepins, also useful synthons for the development of additional novel C2-substituted 3'-deoxyadenosine analogues to be evaluated in development of experimental therapeutics.

Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos -/- and wild-type mice. Inos -/- mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos -/- mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses nonpermissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immuneprivileged sites such as the brain parenchyma, after crossing the blood-brain barrier. In the present study, we have established a protocol for high-purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance.

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The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.