Mauro Shugiro Tada

Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function

Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated “priming,” we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a TLR9-, MyD88-, and IFNγ-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while TLR9−/−, IL12−/− and to a greater extent, IFNγ−/− mice were protected. Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFNγ responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.

Understanding the clinical spectrum of complicated Plasmodium vivax malaria: a systematic review on the contributions of the Brazilian literature

The resurgence of the malaria eradication agenda and the increasing number of severe manifestation reports has contributed to a renewed interested in the Plasmodium vivax infection. It is the most geographically widespread parasite causing human malaria, with around 2.85 billion people living under risk of infection. The Brazilian Amazon region reports more than 50% of the malaria cases in Latin America and since 1990 there is a marked predominance of this species, responsible for 85% of cases in 2009. However, only a few complicated cases of P. vivax have been reported from this region. A systematic review of the Brazilian indexed and non-indexed literature on complicated cases of vivax malaria was performed including published articles, masters dissertations, doctoral theses and national congresses abstracts. The following information was retrieved: patient characteristics (demographic, presence of co-morbidities and, whenever possible, associated genetic disorders); description of each major clinical manifestation. As a result, 27 articles, 28 abstracts from scientific events annals and 13 theses/dissertations were found, only after 1987. Most of the reported information was described in small case series and case reports of patients from all the Amazonian states, and also in travellers from Brazilian non-endemic areas. The more relevant clinical complications were anaemia, thrombocytopaenia, jaundice and acute respiratory distress syndrome, present in all age groups, in addition to other more rare clinical pictures. Complications in pregnant women were also reported. Acute and chronic co-morbidities were frequent, however death was occasional. Clinical atypical cases of malaria are more frequent than published in the indexed literature, probably due to a publication bias. In the Brazilian Amazon (considered to be a low to moderate intensity area of transmission), clinical data are in accordance with the recent findings of severity described in diverse P. vivax endemic areas (especially anaemia in Southeast Asia), however in this region both children and adults are affected. Finally, gaps of knowledge and areas for future research are opportunely pointed out.

Seasonal Malaria Transmission and Variation of Anopheline Density in Two Distinct Endemic Areas in Brazilian Amazônia

Abstract Studies on seasonal anopheline fauna variation were performed in two distinct settlements in the State of Rondônia, Brazil: one at the Madeira River banks (Portuchuelo) with stable native Amazonian population; the other at an inland lumber-extracting farm (Urupá) in dry land, in which adults are mostly migrants. During a 6-yr period (1994–2000), 8,638 adult anophelines were collected: 2,684 in Urupá and 5,954 in Portuchuelo. Anopheles darlingi represented >95% of total mosquitoes caught. Dissection of 4,424 A. darlingi females yielded a very low sporozoite infection index below 0.1%. Oocysts were found in both localities in ∼0.1% of dissected mosquitoes. Determination of the hour biting rates disclosed seasonal variations in both localities. However, in Portuchuelo, mosquito density peaked at the acme of the rainy season, whereas at Urupá it peaked in the dry season. The increase in mosquito density and incidence of malaria cases were coincident. The high mosquito densities observed in the riverine settlement of Portochuelo sector B, which permits evaluation in >10,000 mosquitoes’ bites/person/year, could explain, in spite of the low mosquito’s infection index, the previously described development of natural immunity in the local population that is not observed in the dry land agroindustrial settlement of Urupá.

Neutrophil Paralysis in Plasmodium vivax Malaria

Background The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria. Materials and Methods Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients. Principal Findings Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8). Conclusion Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria.

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14+CD16− (classical), CD14+CD16+ (inflammatory), and CD14loCD16+ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16+ cells, in particular the CD14+CD16+ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14+ were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14+CD16+ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14+CD16+ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Plasmodium falciparum: analysis of transcribed var gene sequences in natural isolates from the Brazilian Amazon region.

Parasite isolates from Brazilian Western Amazonian patients suffering from uncomplicated falciparum malaria were matured in vitro and their var gene transcripts were analysed by RT-PCR and sequencing. Additionally, the cytoadherence patterns of these isolates were determined by panning techniques using transfected CHO cell lines expressing different surface receptors. All of the isolates tested showed between 4 and 13 different var gene transcripts per isolate. Several of these transcripts were present in more than one isolate and three sequences appeared to be preferentially expressed in natural infections. In most of the isolates, cytoadherence occurred to the receptors ICAM-1 and CD36. Several isolates showed a multiadherent profile. Analysis of MSP1 and MSP2 allelic polymorphism indicated polyclonal infections, that could be responsible for the multiadherent phenotype.

Induction of inhibitory receptors on T cells during Plasmodium vivax malaria impairs cytokine production

The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4(+) and CD8(+) T cells. Higher frequencies of CD4(+) express more than 1 regulatory molecule compared to CD8(+) T cells. Moreover, lower proportions of CD4(+) T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin-3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function.

Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections

The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself.

Phenotypic profiling of CD8 + T cells during Plasmodium vivax blood-stage infection

BackgroundFor a long time, the role of CD8+ T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8+ T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8+ T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8+ T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8+ T cells subsets from patients with uncomplicated symptomatic P. vivax malaria.MethodsBlood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann–Whitney test was used depending on the data distribution.ResultsP. vivax-infected subjects had lower percentages and absolute numbers of CD8+CD45RA+ and CD8+CD45RO+ T cells when compared to uninfected individuals (pu2009≤u20090.0002). A significantly lower absolute number of circulating CD8+CD45+CCR7+ cells (pu2009=u20090.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ+ (pu2009=u20090.001), TNF-α+ (pu2009<u20090.0001) and IL-10+ (pu2009<u20090.0001) CD8+ T cells. Despite the reduction in the number of CD8+ memory T cells producing IFN-γ (pu2009<u20090.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8+TNF-α+ (pu2009=u20090.016) and CD8+IL-10+ (pu2009=u20090.004) cells. Positive correlations were observed between absolute numbers of CD8+IL-10+ and numbers of CD8+IFN-γ+ (pu2009<u20090.001) and CD8+TNF-α+ T cells (pu2009≤u20090.0001). Finally, an increase in the plasma levels of TNF-α (pu2009=u20090.017) and IL-10 (pu2009=u20090.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals.ConclusionsP. vivax infection reduces the numbers of different subsets of CD8+ T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8+ memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax.

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.