Visceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus Leishmania. Classic VL is characterized by a systemic infection of phagocytic cells and an intense ...activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with Leishmania (L.) infantum is associated with the development and severity of disease.
Sera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals.
Although differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that L. infantum may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against L. infantum.
Ecto-nucleotidase activity is involved in the infection process of
and various other parasites that enables modulation of host immune responses to promote disease progression. One of the enzymes ...responsible for this activity is the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase). The enzyme hydrolyzes nucleotides tri- and/or di-phosphate into monophosphate products, which are subsequently hydrolyzed into adenosine. These nucleotides can serve as purinergic signaling molecules involved in diverse cellular processes that govern immune responses. Given the importance of the extracellular metabolism of these nucleotides during intracellular pathogen infections, this study evaluates the role of ecto-nucleotidase activity during
(
) infection in human macrophages. E-NTPDase protein expression and activity was evaluated in
during purine starvation, adenosine-enriched medium, or in the presence of an inhibitor of ecto-nucleotidases. Results show that E-NTPDase is expressed in
parasites, including on the cell membrane. Furthermore, functional activity of the enzyme was modulated according to the availability of adenosine in the medium. Purine starvation increased the hydrolytic capacity of nucleotides leading to higher infectivity, while growth in adenosine-enriched medium led to lower infectivity. Moreover, inhibiting E-NTPDase function decreased
infection in macrophages, suggesting the enzyme may serve as a ligand. Taken together, the ability of
to hydrolyze nucleotides is directly associated with increased infectivity in macrophages.
Functionally, cluster of differentiation 14 (CD14) is a co-receptor of the complex formed by lipopolysaccharide (LPS) and LPS-binding protein expressed on the membrane of a variety of cells. However, ...CD14 can be shed from the cell membrane into the circulation as soluble CD14 (sCD14) upon cell activation. Previously, our group reported that elevated sCD14 serum levels were associated with the clinical and laboratory findings in the context of visceral leishmaniasis (VL), but not in the context of LPS stimulation or bacterial infection. In the present study, we investigated the secretion dynamics of sCD14 in the context of
Leishmania infantum (syn. L. chagasi)
in vitro infection. Macrophages from treated VL patients and delayed-type hypersensitivity positive (DTH
+
) subjects were infected with
L. infantum (syn. L. chagasi)
promastigotes, and the infection index was evaluated (number of amastigotes per 100 infected macrophages). Additionally, the levels of sCD14, Inteleukin (IL)10, IL-6 and tumour necrosis factor alpha (TNF-α) were measured in the culture supernatants using the Luminex assay. Interestingly, the release of sCD14 was inversely correlated with the
L. infantum (syn. L. chagasi)
infection index. Of note, the release of sCD14 was upregulated and downregulated in the context of infected macrophages from DTH
+
subjects and treated VL patients, respectively. Additionally, we also observed that the levels of sCD14 in the culture supernatants were positively correlated with the levels of TNF-α, IL-6 and IL-10. Therefore, our data suggest that macrophages from treated VL patients and DTH
+
subjects respond differently to
L. infantum
(
syn. L. chagasi
) infection in the context of the release of sCD14; therefore, the release of sCD14 may be associated with the outcome of VL.
This work describes the heterologous expression and enzymatic activity of NTPDase-1, which is ubiquitously expressed in epimastigotes, participating in the T. cruzi adhesion to host cells and ...infection.
•Heterologous TcNTPDase-1 showed a greater capacity to hydrolyze NDPs than NTPs.•TcNTPDase1 Km for ATP would permit it to influence host purinergic signaling.•TcNTPDase-1 showed a ubiquitous localization in epimastigotes.•TcNTPDase-1 may be involved in a number of different biological processes.•TcNTPDase-1 acts as a pro-adhesion molecule.
Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite–host cell adhesion. These data open new frontiers to future studies on this specific parasite–host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.
IntroductionZika virus (ZIKV) infection during pregnancy is a known cause of microcephaly and other congenital and developmental anomalies. In the absence of a ZIKV vaccine or prophylactics, ...principal investigators (PIs) and international leaders in ZIKV research have formed the ZIKV Individual Participant Data (IPD) Consortium to identify, collect and synthesise IPD from longitudinal studies of pregnant women that measure ZIKV infection during pregnancy and fetal, infant or child outcomes.Methods and analysisWe will identify eligible studies through the ZIKV IPD Consortium membership and a systematic review and invite study PIs to participate in the IPD meta-analysis (IPD-MA). We will use the combined dataset to estimate the relative and absolute risk of congenital Zika syndrome (CZS), including microcephaly and late symptomatic congenital infections; identify and explore sources of heterogeneity in those estimates and develop and validate a risk prediction model to identify the pregnancies at the highest risk of CZS or adverse developmental outcomes. The variable accuracy of diagnostic assays and differences in exposure and outcome definitions means that included studies will have a higher level of systematic variability, a component of measurement error, than an IPD-MA of studies of an established pathogen. We will use expert testimony, existing internal and external diagnostic accuracy validation studies and laboratory external quality assessments to inform the distribution of measurement error in our models. We will apply both Bayesian and frequentist methods to directly account for these and other sources of uncertainty.Ethics and disseminationThe IPD-MA was deemed exempt from ethical review. We will convene a group of patient advocates to evaluate the ethical implications and utility of the risk stratification tool. Findings from these analyses will be shared via national and international conferences and through publication in open access, peer-reviewed journals.Trial registration numberPROSPERO International prospective register of systematic reviews (CRD42017068915).
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