Paulo Renato Rivas Totino

Plasmodium falciparum: erythrocytic stages die by autophagic-like cell death under drug pressure.

It has been reported that an apoptotic cell death process can occur with protozoans, but no consensus on Plasmodium susceptibility to apoptosis was reached till now. Thus, we evaluated if Plasmodium falciparum blood forms undergo apoptosis after in vitro pressure with chloroquine, S-nitroso-N-acetyl-penicillamine (SNAP) or staurosporine. Inhibition of parasite growth and loss of viability were observed in treated cultures by both light microscopy and flow cytometry. When DNA fragmentation was verified, only a small number of TUNEL-positive parasites was detected in treated cultures and pretreatment of parasite with a general caspase inhibitor was not able to prevent parasite death. Considering the lack of apoptotic characteristics and the observation of parasites with cytoplasmatic vacuolization by electron microscopy, we conclude that P. falciparum parasites under chloroquine, SNAP or staurosporine pressures do not die by apoptosis but by a process similar to autophagy. The autophagic pathway could be explored as an alternative target for the development of new antimalarial drugs.

Dexamethasone has pro-apoptotic effects on non-activated fresh peripheral blood mononuclear cells.

Apoptosis is a physiological method of cell death commonly referred to as programmed cell death. However, non‐apoptotic programmed cell death, such as autophagy and programmed necrosis, has been characterized by morphological criteria. In view of the human therapeutic use of DEX, and considering that no difference in the number and/or affinity of glucocorticoid receptors in activated and non‐activated lymphocytes has been reported, we decided to evaluate the effect of DEX on fresh peripheral blood mononuclear cells (PBMC). Transmission electron microscopy showed that DEX can significantly induce apoptosis in non‐activated PBMC. It was also observed by transmission electron microscopy that, independently of DEX treatment, PBMC also died by a process marked by extreme vacuolization and increase in cellular volume; these cells were erroneously classified as viable by flow cytometry using the 7‐AAD assay. It is concluded that the DEX pro‐apoptotic effect is not restricted to activated PBMC and, therefore, DEX‐induced apoptosis could play either homeostatic (activated PBMC) or immunosuppressive (non‐activated PBMC) roles.

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.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Pro-apoptotic effects of antimalarial drugs do not affect mature human erythrocytes

Malaria is an important public health problem worldwide, representing also an obstacle for the development of the countries, mainly in the African continent. Since no effective vaccine has been developed yet, early diagnosis and prompt treatment are the main strategy to control malaria transmission. Many of the drugs used for malaria treatment have the ability to induce apoptosis in different cell types. In addition, apoptosis has also been identified in enucleated cells. The present work is aimed, therefore, to evaluate the pro-apoptotic aptness of chloroquine, quinine, artemisinin and mefloquine on mature erythrocytes by flow cytometry through the detection of cell shrinkage and phosphatidylserine exposure at the cell surface-hallmarks of apoptosis. Although we observed that known apoptosis inducer, such as ionomycin, had led to erythrocyte apoptosis, we were not able to detect any pro-apoptotic effect of the studied antimalarial drugs on these cells. We conclude that chloroquine, quinine, artemisinin and mefloquine may not be able to induce apoptosis in erythrocytes and, therefore, do not seem to contribute to malaria associated erythrocyte destruction and anemia.

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

Plasmodium falciparum , but not P. vivax , can induce erythrocytic apoptosis

BackgroundApoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections.FindingsApoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells.ConclusionsApoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

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.

Evidencing the Role of Erythrocytic Apoptosis in Malarial Anemia

In the last decade it has become clear that, similarly to nucleated cells, enucleated red blood cells (RBCs) are susceptible to programmed apoptotic cell death. Erythrocytic apoptosis seems to play a role in physiological clearance of aged RBCs, but it may also be implicated in anemia of different etiological sources including drug therapy and infectious diseases. In malaria, severe anemia is a common complication leading to death of children and pregnant women living in malaria-endemic regions of Africa. The pathogenesis of malarial anemia is multifactorial and involves both ineffective production of RBCs by the bone marrow and premature elimination of non-parasitized RBCs, phenomena potentially associated with apoptosis. In the present overview, we discuss evidences associating erythrocytic apoptosis with the pathogenesis of severe malarial anemia, as well as with regulation of parasite clearance in malaria. Efforts to understand the role of erythrocytic apoptosis in malarial anemia can help to identify potential targets for therapeutic intervention based on apoptotic pathways and consequently, mitigate the harmful impact of malaria in global public health.

Modulation of cytochrome P450 2A5 activity by lipopolysaccharide: low-dose effects and non-monotonic dose-response relationship.

Mouse cytochrome P450 (CYP) 2A5 is induced by inflammatory conditions and infectious diseases that down-regulate the expression and activity of most other CYP isoforms. Enhanced oxidative stress and nuclear factor (erythroid 2-related factor) 2 (Nrf2) transcription factor activation have been hypothesised to mediate up-regulation of CYP2A5 expression in the murine liver. The unique and complex regulation of CYP2A5, however, is far from being thoroughly elucidated. Sepsis and high doses of bacterial lipopolysaccharide (LPS) elicit oxidative stress in the liver, but depression, not induction, of CYP2A5 has been observed in studies of mice treated with LPS. The foregoing facts prompted us to evaluate the response of CYP2A5 liver activity in female DBA-2 mice over a broad range of LPS doses (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 mg/kg). Cytokine levels (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon gamma, tumour necrosis factor alpha) and nitric oxide (NO) were measured in the blood serum. Activities of CYP1A (EROD) and CYP2B (BROD) in the liver were also determined for comparative purposes. LPS depressed CYP2A5 at low doses (0.025–2.0 mg/kg) but not at doses (>2 mg/kg) that increased pro-inflammatory cytokines and NO serum levels, and depressed CYP1A and CYP2B activities. Blockade of pro-inflammatory cytokines and the overproduction of NO induced by co-treatment with pentoxifylline and LPS and iNOS inhibition with aminoguanidine both extended down-regulation of CYP2A5 to the high dose range while not affecting LPS-induced depression of CYP1A and CYP2B. Overall, the results suggested that NO plays a role in the reversal of the low-dose LPS-induced depression of CYP2A5 observed when mice were challenged with higher doses of LPS.