Refractoriness of Eryptotic Red Blood Cells to Plasmodium falciparum Infection: A Putative Host Defense Mechanism Limiting Parasitaemia

Recently, we have described that apoptosis-like process of red blood cells (RBC) – eryptosis – in malaria is not restricted to parasitized cells, occurring also in non-parasitized RBC (nRBC). Besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation. In the present study, we investigated the capacity of P. falciparum parasites to infect eryptotic RBC. Schizont parasitized RBC concentrated by magnetic separation were cultured with eryptotic RBC obtained by ionomycin treatment and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. While parasites infected and developed normally in control non-eryptotic RBC, cultures performed with eryptotic RBC had a marked decrease in parasitaemia. It was noteworthy a great number of free merozoites in eryptotic RBC cultures, indicating that these cells were not susceptible to invasion. We suggest that although eryptosis could be involved in malaria pathogenesis, it could also acting protectively by controlling parasite propagation

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice’s splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine

Apoptose em eritrócitos: envolvimento na patogênese da anemia da malária

Submitted by Alessandra Portugal ([email protected]) on 2013-09-17T15:01:12Z No. of bitstreams: 1 Tese Paulo Totino 2012.pdf: 4009343 bytes, checksum: 0cf12e4e6c9631d541933a237d4cea87 (MD5)Made available in DSpace on 2013-09-17T15:01:12Z (GMT). No. of bitstreams: 1 Tese Paulo Totino 2012.pdf: 4009343 bytes, checksum: 0cf12e4e6c9631d541933a237d4cea87 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A anemia grave é sem dúvida uma das complicações mais comuns durante a infecção malárica e é uma das principais responsáveis pela elevada taxa de mortalidade de crianças e mulheres grávidas em regiões hiperendêmicas africanas. Embora os mecanismos que desencadeiam a anemia grave da malária ainda não tenham sido completamente elucidados, sabe-se que o aumento da destruição dos eritrócitos não parasitados tem importante contribuição na gênese dessa manifestação clínica. Frente a esse fato e tendo em vista a já descrita susceptibilidade dos eritrócitos ao processo de apoptose, que inclusive parece estar relacionado com a anemia de diversas patologias e pode ser induzida por diferentes tipos de drogas, decidimos estudar a apoptose eritrocitária e sua participação na anemia no decurso da infecção malárica. Primeiramente, avaliamos in vitro a capacidade da cloroquina, mefloquina, artemisinina e quinina em induzir apoptose em eritrócitos humanos. Nesta primeira investigação, observamos que os antimaláricos não tiveram efeito proapoptótico nestas células e, portanto, parecem não participar da anemia da malária através da indução de apoptose eritrocitária. Em seguida, estudando a infecção experimental de camundongos Balb/c pelo Plasmodium yoelii 17XL, constatamos um aumento nos níveis basais de apoptose de eritrócitos não parasitadas e sugerimos que este aumento poderia estar contribuindo com a anemia neste modelo experimental de malária. Porém, quando posteriormente examinamos em paralelo os níveis de apoptose e o grau de anemia, não foi possível observar relação entre estes dois eventos, mas sim entre a carga parasitária e os níveis de apoptose detectados na fase tardia da infecção. A possível influência da parasitemia nos níveis de apoptose eritrocitária parece ter encontrado suporte quando estudamos pacientes infectados pelo P. vivax. De fato, nessa infecção, que cursa com baixa parasitemia, não detectamos alteração nos níveis de apoptose eritrocitária. Como a apoptose além de ter propriedades patogênicas, também está envolvida com a limitação da propagação de parasitas intracelulares, avaliamos então in vitro a capacidade do P. falciparum de infectar eritrócitos apoptóticos. Neste último estudo, verificamos que estas células apoptóticas são refratárias à infecção plasmodial e, portanto, poderiam participar no controle da parasitemia na malária.Severe anaemia is a common complication associated to malaria and plays an important role on mortality, particularly in children and pregnant woman living in malaria hyperendemic African regions. It is well known that together to other mechanisms leading to severe malaria anaemia, premature elimination of non-parasitized red blood cells (nRBC) contributes to the genesis of this malarial complication. Thus, considering the susceptibility of RBC to apoptosis – process that has been related to the anaemia of several pathologies and that can be induced by different drugs – we decided to study erythrocytic apoptosis and its involvement in malaria-associated anaemia. Firstly, we evaluated the in vitro capacity of chloroquine, quinine, artemisinin and mefloquine to induce apoptosis in human RBC. In this first study, it was observed that antimalarial drugs were not able to induce apoptosis in RBC and, therefore, it seems that these drugs do not participate in malaria anameia through induction of erythrocytic apoptosis. Next, studying the experimental infection of P. yoelii 17XL in Balb/c mice, we noted increased levels of nRBC apoptosis that could be related to anaemia in this malaria model. However, when we analyzed, in parallel, apoptotic levels and anaemia degree, no relationship between these two parameters was observed, although an association between parasite load and apoptosis at the later stages of infection had been observed. The putative influence of parasitaemia on apoptotic levels seems to be supported by our studies with P. vivax malaria patients, since the low parasitaemia observed in this infection was not followed by an increase of erytrocytic apoptosis. Considering that besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation, we also investigated if P. falciparum parasites were able to infect apoptotic RBC. In this last study, it was possible to observe that apoptotic RBC were refractory to in vitro plasmodial infection and, therefore, it could act protectively by controlling malaria parasite propagation

Effect of ionomycin in <i>P. falciparum</i> parasite growth.

<p>Asynchronous cultures (0 h) were maintained for 18 h in presence of ionomycin or lysates of non-treated (nL) or ionomycin-treated (iL) RBC and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. Non-treated cultures were used as control. Data represent mean±S.D. of parasitaemia of the cultures performed in fiveplicate. *** indicates significant difference (p<0.001; ANOVA) from initial culture (0 h).</p

Resistance of eryptotic RBC to <i>P. falciparum</i> infection.

<p>Schizont pRBC concentrated by magnetic separation were cultured with control non-eryptotic RBC (normal RBC) or eryptotic RBC and, then, parasites invasion (16 and 60 h) and development (36 h) were evaluated in Giemsa-stained thin blood smears. (A) Mean±S.D. of parasitaemia and (B) representative photomicrographs (scale bar  =  20 µm) of the cultures performed in fiveplicate showing: i) eryptotic RBC are refractory to merozoite invasion (16 h); ii) eryptotic RBC are smaller than normal RBC (0–60 h) iii) parasite development in eryptotic RBC culture take place only into normal size RBC (36–60 h) and; iv) eryptotic RBC were still detected after 60 hours of culture. *** indicates significant difference (p<0.001; ANOVA) from initial culture (0 h) in (A) and arrows indicate free merozoites in (B).</p

Induction of RBC eryptosis.

<p>RBC (A, R1) were incubated at 37°C for 4 h at 2% hematocrit in presence (gray) or absence (black line) of ionomycin (1 µM) and, then, induction of eryptosis was verified by forward scatter measurement (B) and annexin V-PE staining (C).</p

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis

Modulation of Signal Regulatory Protein &alpha; (SIRP&alpha;) by Plasmodium Antigenic Extract: A Preliminary In Vitro Study on Peripheral Blood Mononuclear Cells

Signal regulatory protein &alpha; (SIRP&alpha;) is an immunoreceptor expressed in myeloid innate immune cells that signals for inhibition of both phagocytosis and inflammatory response. Malaria parasites have evolutionarily selected multiple mechanisms that allow them to evade host immune defenses, including the modulation of cells belonging to innate immunity. Notwithstanding, little attention has been given to SIRP&alpha; in the context of immunosuppressive states induced by malaria. The present study attempted to investigate if malaria parasites are endowed with the capacity of modulating the expression of SIRP&alpha; on cells of innate immune system. Human peripheral blood mononuclear cells (PBMC) from healthy individuals were incubated in the presence of lipopolysaccharide (LPS) or crude extracts of P. falciparum or P. vivax and then, the expression of SIRP&alpha; was evaluated by flow cytometry. As expected, LPS showed an inhibitory effect on the expression of SIRP&alpha; in the population of monocytes, characterized by cell morphology in flow cytometry analysis, while Plasmodium extracts induced a significant positive modulation. Additional phenotyping of cells revealed that the modulatory potential of Plasmodium antigens on SIRP&alpha; expression was restricted to the population of monocytes (CD14+CD11c+), as no effect on myeloid dendritic cells (CD14&minus;CD11c+) was observed. We hypothesize that malaria parasites explore inhibitory signaling of SIRP&alpha; to suppress antiparasitic immune responses contributing to the establishment of infection. Nevertheless, further studies are still required to better understand the role of SIRP&alpha; modulation in malaria immunity and pathogenesis