Cellular and humoral immune responses against the Plasmodium vivax MSP-1(19) malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Background: Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.Methods: the study was carried out in Paragominas, Para State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-1(19)) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-gamma and IL-10 were measured by enzyme-linked immunosorbent assay.Results: the prevalence of activated CD4(+) was greater than CD8(+) T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-1(19) and PSS1 antigen. A low proliferative response against PvMSP-1(19) and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-gamma and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-1(19) stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient's cells while low levels of IFN-gamma and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-1(19) was evidenced, regardless class or IgG subclass. PvMSP-1(19)-induced antibodies were predominantly on non-cytophilic subclasses.Conclusions: the results presented here shows that PvMSP-1(19) was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-1(19) in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Oswaldo Cruz (FIOCRUZ, Brazil)Fiocruz MS, Inst Oswaldo Cruz, Lab Pesquisas Malaria, BR-21040900 Rio de Janeiro, RJ, BrazilFiocruz MS, Ctr Pesquisa Diagnost & Treinamento Malaria CPD M, Reference Ctr Malaria Extra Amazonian Reg Secreta, BR-21040900 Rio de Janeiro, RJ, BrazilFiocruz MS, Inst Oswaldo Cruz, Lab Biol Mol & Doencas Endem, BR-21040900 Rio de Janeiro, RJ, BrazilUniv São Paulo, Dept Analises Clin & Toxicol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilSVS, Inst Evandro Chagas, Programa Ensaios Clin Malaria, Belem, Para, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilWeb of Scienc

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract\ud \ud \ud \ud Background\ud \ud Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.\ud \ud \ud \ud Methods\ud The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay.\ud \ud \ud \ud Results\ud The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses.\ud \ud \ud \ud Conclusions\ud The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.We are grateful to all patients that agreed to participate in this study for their cooperation and generous donation of blood, which made this study possible. This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Instituto Oswaldo Cruz (FIOCRUZ, Brazil). MFFC and CTDR are recipients of a Research Productivity Fellowship from CNPq and receive a grant from FAPERJ as "Cientistas do Nosso Estado"

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

Submitted by sandra infurna ([email protected]) on 2016-04-18T14:25:37Z No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-18T14:46:01Z (GMT) No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5)Made available in DSpace on 2016-04-18T14:46:01Z (GMT). No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.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 proinflammatory 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

Concentrations of IL-2, IL-4, and IL-10 in the blood serum of mice treated with PTX and LPS.

<p>Concentrations of cytokines were measured in the serum of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg i.p., N = 8 per dose group [except the 0 and 20 mg/kg LPS groups, N = 10]) alone (●) or with PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per LPS dose group) (▲). Data are shown as means ± SEM: <b>a</b> indicates that the mean differs from control value in the LPS-treated group; <b>b</b> indicates that the mean differs from control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from the mean value in the LPS-treated group for the same dose (Kruskal-Wallis and Mann-Whitney tests, P < 0.05).</p

Levels of pro-inflammatory cytokines in the blood serum of mice treated with pentoxifylline (PTX) and LPS.

<p>Concentrations of tumour necrosis factor alpha (TNF-α), interferon gamma (IFN- γ), interleukin (IL)-6, and IL-17A were measured in the serum of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg i.p., N = 8 per dose group (except the 0 and 20 mg/kg LPS groups, N = 10) alone (●) or PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per dose group) (▲). Data represent means ± SEM: <b>a</b> indicates that the mean differs from the control value in the LPS-treated group; <b>b</b> indicates that the mean differs from the control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from that in the LPS-treated group for the same dose (Kruskal-Wallis and Mann-Whitney tests, P < 0.05).</p

Activities (pmol/[min·mg protein]) of CYP2A5 (COH) and CYP1A and CYP2B (EROD and BROD, respectively) in the liver of mice treated with PTX and LPS.

<p>Monooxygenase activities were measured in the liver of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg, i.p., N = 10 per dose group) alone (●) or with PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per LPS dose group) (▲). Data are shown as means ± SEM: <b>a</b> indicates that the mean differs from control value in the LPS-treated group; <b>b</b> indicates that the mean differs from control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from the mean value in the LPS-treated group for the same dose (ANOVA and Dunnett’s multiple comparison test followed by the Student’s <i>t</i>-test, P < 0.05).</p

Non-monotonic dose response of CYP2A5 activity in mouse livers after treatment with LPS.

<p>NO concentration (μM) and COH activity (pmol/[min·mg protein]) in the hepatic microsomal fraction of liver samples from female DBA-2 mice 24 h after treatment. Mice per LPS dose (mg/kg i.p.) group numbered as follows: 0 (PBS), N = 26; 0.025, N = 3; 0.05, N = 8; 0.1, N = 6; 0.2, N = 6; 0.5, N = 8; 1, N = 6; 2, N = 7; 5, N = 7; 10, N = 10; and 20, N = 7. Data represent means ± SEM. An asterisk (<b>*</b>) indicates that the mean value differs (ANOVA and Dunnett’s multiple comparison test, P < 0.05) from that of the control group.</p

Changes in nitric oxide (NO) concentration in the blood serum and cytochrome P450 (CYP) 2A5, CYP1A, and CYP2B activities in the liver with time elapsed after lipopolysaccharide (LPS) injection.

<p>Nitrite concentration (μM), coumarin 7-hydroxylase (COH, CYP2A5), benzyloxy-resorufin-<i>O</i>-debenzylase (BROD, CYP2B), and ethoxy-resorufin-<i>O</i>-deethylase (EROD, CYP1A) activities (pmol/[min·mg protein]) in the hepatic microsomal fraction of liver samples from female DBA-2 mice injected intraperitoneally (i.p.) with phosphate-buffered saline (control) or LPS (2, 5 or 20 mg/kg) were determined 6, 12 or 24 h after treatment. N = 7 for all groups, except for the control (0 h; N = 20) and LPS (5 mg/kg) groups at 6 h (N = 15). Data represent means ± standard error of the mean (SEM). An asterisk (<b>*</b>) indicates that the mean value differs (analysis of variance [ANOVA] and Dunnett’s multiple comparison test, P < 0.05) from that of the control group.</p

Effect of inducible NO synthase (iNOS) inhibition with aminoguanidine (AG) on LPS-induced changes in CYP2A5 (COH) and CYP1A and CYP2B (EROD and BROD, respectively) activities (pmol/[min·mg protein]) in the mouse liver.

<p>NO levels were determined in the blood serum and monooxygenase was measured in liver microsomes from female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0 or 10 mg/kg i.p.) alone or LPS (0 or 10 mg/kg i.p.) plus AG (0, 50 or 100 mg/kg i.p.). The mice per group numbered as follows: 0 (PBS alone), N = 7; LPS alone, N = 9; a50 (AG, 50 mg/kg plus PBS), N = 6; a100 (AG, 100 mg/kg plus PBS), N = 6; a50L (AG, 50 mg/kg plus LPS), N = 6; and a100L (AG, 100 mg/kg plus LPS), N = 6. Data are shown as means ± SEM. <b>a</b> and <b>b</b> above the bars indicate that the mean values differ (ANOVA and Bonferroni’s multiple comparison test, P < 0.05) from that of the group treated with PBS alone (a), LPS alone (b), or both.</p