Análise genômica comparativa e os polimorfismos nos genes TNFA, IFNG IL6 e IL10 associados à expressão de citocinas na infecção por Plasmodium vivax no município do Itaituba, Estado do Pará

In endemic areas of Asia, Oceania, Central and South America and in the horn of Africa P. vivax malaria is a major cause of morbidity with 35 million cases annually. In Brazil, the Amazon region concentrates almost all cases and infections registered countrywide, with more than three hundred thousand cases per year. Several evidences suggest that an exacerbated inflammatory response associated to density parasite is likely to aggravate the malaria symptoms. We assessed the haematological and immunological aspects, genetic alterations related to CNVs that could lead to phenotypic alterations, conferring resistance or susceptibility to malaria, as well the presence of polymorphisms in cytokine genes and their association with the infection in patients living in a gold-mining area in a gold-mining in the Brazilian Amazon Region, establishing patterns of immune response characteristic of primary malaria, recurrent malaria and endemic control. Six SNPs (TNFA-308G/A, IFNG+874T/A, IL6-174G/C, IL10-1082G/A, -819C/T, -592C/A) in four genes were determined; blood cell count was conducted on automatic analyzer; plasmatic cytokines IL-6, IL-10, TNF-α and IFN-γ were quantified by flow cytometry and density parasite was estimated by thick blood films with confirmation by nested-PCR; the CNV was estimated by aCGH and association between copy number and phenotypes (parasite load, mean number of clinical infections of malaria and gender) was assessed. The statistical analyzes were performed by Graph-pad prism 6.0 and Bioestat 5.0. No significant association was found between SNPs and malaria infection; cytokine levels were higher in malaria group when compared to endemic control; production of IL-10 was higher in the presence of GCC/GCC haplotype; IFN-γ levels were correlated with previous malaria episodes; malaria patients showed lower platelet numbers, reduction on white blood cells count and an increased monocyte percentage; significant increase in the IL-6 and IL-10 plasmatic levels in both malaria groups; the primary malaria patients displayed the highest significant plasmatic IFN-γ levels; recurrent malaria patients displayed the highest significant plasmatic TNF-α; malaria infection demonstrated correlation between parasite density and TNF-α, IL-6 and IL-10 levels; a total of 112 amplified genes and 12 deleted genes were observed and the CNVs found did not include any gene related to receptors or vivax malaria resistance factors. There were no statistically significant correlations between the clinical and pathological data (parasite load, mean number of clinical infections of malaria and gender) and the presence of CNVs in the patients studied. This study provides additional data on Plasmodium-host immune response and describes the quantitative changes in the human genome in P. vivax infection in an endemic area of garimpo.A malária é uma das mais importantes doenças transmitidas por vectores e que acomete grande parte da população mundial. O P. vivax é a espécie predominante nas Américas, representando 64% dos casos. A área endêmica da malária no Brasil compreende a Região Amazônica, responsável por 99% dos casos autóctones. Trabalhos anteriores demonstraram a alta prevalência da malária em garimpeiros na região Amazônica e a espécie mais frequente envolvida é o P. vivax. A resistência à malária faz parte da interação de diferentes fatores que regulam as relações entre o parasito e o hospedeiro. Essa interação é um fator determinante na resposta imune a infecção. Os polimorfismos de nucleotídeos únicos (SNPs) nos genes de citocinas têm sido explorados em estudos de variação genética humana e tentam explicar a influência dessa variação na susceptibilidade as doenças infecciosas. As variações no número de cópias (CNVs) também podem afetar potencialmente a expressão dos genes de várias maneiras, contudo pouco se sabe sobre o papel dessa variação genética na adaptação humana frente à infecção pelo P. vivax. O estudo objetivou investigar se as alterações genômicas quantitativas e os SNPs nos genes TNFA-308A>G, IFNG+874T>A, IL6-174C>G e do haplótipo -1082/-592/-819 no gene IL10, associados à produção de citocinas podem influenciar na fisiopatogenia da malária vivax. O estudo selecionou 129 pacientes, a partir de 288 atendidos na cidade de Itaituba, estado do Pará, sendo 79 pacientes sem infecção para malária e 50 pacientes positivos para P. vivax e destes, 15 tinham infecção primária a malária e 35 pacientes relataram episódios anteriores a infecção. Foi determinada a carga parasitária pela gota espessa; os parâmetros hematológicos; as citocinas foram dosas por citometria de fluxo; a genotipagem dos SNPs foi feita por PCR-SSP e a variação no número de cópias foi determinada pela técnica do aCGH. A análise estatística foi realizada utilizando o programa Bioestat e Graph-pad prim. Observados que ambos os grupos maláricos (primário e recorrente) apresentaram redução significativa no número de plaquetas; no grupo recorrente observamos aumento significativo na porcentagem de monócitos; não observamos diferenças significativas na frequência dos SNPs; não observamos associações dos SNPs com a infecção pelo P. vivax; observamos diferenças significativas nos níveis plasmáticas das citocinas entre os grupos; observamos níveis significativamente maiores nos indivíduos com o haplótipo IL10GCC/GCC e correlação entre os níveis das citocinas IL-10, TNF-α e IL-6 e a carga parasitária; observamos um aumento significativo no nível das citocinas IFN-γ, IL-6 e IL-10 no grupo com malária primária em comparação ao controle endêmico, bem como um aumento significativo na expressão das citocinas TNF-α, IL-6 e IL-10 no grupo recorrente em comparação ao grupo controle, enquanto que a expressão da citocina IFN-γ foi significativamente maior no grupo primário em comparação com o grupo recorrente. Descrevemos pela primeira as alterações quantitativas no genoma dos pacientes infectados com P. vivax e identificamos 112 genes amplificados e 12 genes deletados na população em estudo, e, apesar das CNVs encontradas não incluírem nenhum gene relacionado com receptores ou fatores de resistência a malária vivax e nem estarem correlacionados com os dados clinico-patológicos da doença, observamos vários genes com alterações no número de cópias relacionados a outras doenças. Este estudo fornece dados adicionais sobre a resposta imune entre P. vivax e o hospedeiro e as alterações genômicas quantitativas no hospedeiro de uma área endêmica de garimpo.Instituto Evandro Chaga

Granulocyte macrophage colony-stimulating factor alone reduces Toxoplasma gondii replication in microglial culture by superoxide and nitric oxide, without IFN-γ production: a preliminary report

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Federal University of Pará. Postgraduate Program in Neuroscience and Cell Biology. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microscopia eletrônica. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microscopia eletrônica. Ananindeua, PA, Brasil.Federal University of Pará. Institute of Biological Sciences. University Hospital João de Barros Barreto. Laboratory of Investigations in Neurodegeneration and Infection. Belém, PA, Brazil.State University of North Fluminense. Center of Biosciences and Biotechnology. Laboratory of Cell and Tissue Biology. Goytacazes, RJ, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microscopia eletrônica. Ananindeua, PA, Brasil.In vitro studies have demonstrated that GM-CSF in combination with other stimulatory factors induces a microbicidal response that control T. gondii infection. We assessed whether GM-CSF alone can control T. gondii replication in murine microglial cultures. Microglia were collected and cultured with or without GM-CSF and the half of each group was infected with T. gondii. We determined the T. gondii infectivity, cytokines levels, NO and superoxide detection. GM-CSF alone primes microglia, which after infection induces the production of TNF-α and IL-6, leading to NO and superoxide production, without any stimulus from IL-12p70 and IFN-γ

Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and other inflammatory mediators in malaria by Plasmodium vivax during enteroparasites coinfection

This work was supported by National Council for Scientific and Technological Development of Brazil (CNPq) (MCTI/CNPQ/28/ 2018: 434623/2018-0 to TRdeM) (306767/ 2018-0 to RLDM). Support Foundation for Research and Technological Innovation of the State of Sergipe (FAPITEC) (MS/CNPq/FAPITEC/SE/SES/850226/ 2017: 019.203.00933/2018-0 to TRdeM) www.fapitec.se.gov.br. Research Support Foundation of the State of Rio de Janeiro, Brazil-FAPERJ (26/202.808/2017-232679 to RLDM) http://www.faperj.br/Universidade Federal Fluminense, Niterói. Instituto Biomédico. Programa de Pós-Graduação em Microbiologia e Parasitologia Aplicadas. Rio de Janeiro, RJ, Brasil.Universidade Federal de Sergipe. Programa de Pós-Graduação em Biologia Parasitária. São Cristóvão, SE, Brasil.Universidade Federal do Amapá. Departamento de Ciências Biológicas e da Saúde. Macapá, AP, Brasil.Superintendência de Vigilância em Saúde do Estado do Amapá. Macapá, AP, Brasil.Federal University of Sergipe. Health Sciences Graduate Program. São Cristóvão, SE, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal Fluminense, Niterói. Instituto Biomédico. Programa de Pós-Graduação em Microbiologia e Parasitologia Aplicadas. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense, Niterói. Instituto Biomédico. Programa de Pós-Graduação em Microbiologia e Parasitologia Aplicadas. Rio de Janeiro, RJ, Brasil / Universidade Federal de Sergipe. Programa de Pós-Graduação em Biologia Parasitária. São Cristóvão, SE, Brasil.Universidade Federal de Sergipe. Programa de Pós-Graduação em Biologia Parasitária. São Cristóvão, SE, Brasil / Federal University of Sergipe. Health Sciences Graduate Program. São Cristóvão, SE, Brazil / Universidade Federal de Sergipe. Centro de Ciências Biológicas e da Saúde. Departamento de Morfologia. São Cristóvão, SE, Brasil.Universidade Federal de Sergipe. Programa de Pós-Graduação em Biologia Parasitária. São Cristóvão, SE, Brasil / Universidade Federal de Sergipe. Centro de Ciências Biológicas e da Saúde. Departamento de Biologia. São Cristóvão, SE, Brasil.Malaria is a major health issue with more than 200 million cases occurring annually. Moreover, in Malaria endemic area are frequently observed Malaria-enteroparasite co-infections associated with the modulation of inflammatory response. In this aspect, biomarkers play an important role in the disease prognosis. This study aimed to evaluate inflammatory mediators in malaria during coinfection with enteroparasites. A subset of serum samples already collected was analyzed and divided into four groups: Malaria (n = 34), Co-infected (n = 116), Enteroparasite (n = 120) and Control (n = 95). The serum levels of sTREM-1 and IL-6 were measured by ELISA. TNF-α, and IL-10 levels were previously carried out by flow cytometry. Higher serum levels of sTREM-1 and IL-6 were showed in malaria patients compared to healthy controls. In co-infected malarial patients sTREM-1 serum levels were similar to control group. Interestingly, co-infected malaria patients showed IL-6 serum levels decreased compared to individuals only infected with P. vivax. However, in Malaria patients and co-infected there was a positive correlation between the IL-6 and IL-10 levels (P < 0.0001). This is the first report of sTREM-1 levels in P. vivax infected. Moreover, the results revealing a divergent effect of co-infection with the increased balance between pro-and anti-inflammatory cytokines and reduced IL-6 levels but increases the anemia occurrence. The results also highlight the potential use of IL-6 as a biomarker for P. vivax and enteroparasites coinfection

Polymorphism in the IL-1β promoter is associated with IgG antibody response to circumsporozoite protein repeats of Plasmodium vivax

Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES – DS and PDSE) – Finance Code 001São Paulo State University. Graduate Program in Biosciences. São José do Rio Preto, SP, Brazil.Lisboa University. Tropical Medicine and Hygiene Institut. Global Health and Tropical Medicine. Lisboa, Portugal.Federal University of Sergipe. Department of Biology. Aracajú, SE, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Imunogenética da Malária Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microscopia Eletrônica. Ananindeua, PA, Brasil / Universidade Federal do Pará. Belém, PA, Brasil.Fluminense Federal University. Center of Microorganisms Investigation. Niterói, RJ, Brazil.Fluminense Federal University. Center of Microorganisms Investigation. Niterói, RJ, Brazil.Fluminense Federal University. Center of Microorganisms Investigation. Niterói, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Malaria Research. Rio de Janeiro, RJ, Brazil.São Paulo State University. Graduate Program in Biosciences. São José do Rio Preto, SP, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.São Paulo State University. Graduate Program in Biosciences. São José do Rio Preto, SP, Brazil / Fluminense Federal University. Center of Microorganisms Investigation. Niterói, RJ, Brazil.BACKGROUND: It is well established that infection by Plasmodium vivax is a result of host-parasite interactions. In the present study, association with the IL1/IL2 cytokine profiles, anticircumsporozoite protein antibody levels and parasitic loads was evaluated in individuals naturally infected with P. vivax in an endemic area of the Brazilian Amazon. METHODS: Molecular diagnosis of P. vivax and variants was performed using the PCR-RFLP method and IL1B -511C>T, IL2 -330T>G and IL2+114T>G polymorphisms were identified using PCR-RFLP and allele-specific PCR. IL-1β and IL-2 cytokine levels were detected by flow cytometry and circumsporozoite protein (CSP) antibodies were measured by ELISA. RESULTS: Three variants of P. vivax CSP were identified and VK247 was found to be the most frequent. However, the prevalence and magnitude of IgG antibodies were higher for the VK210 variant. Furthermore, the antibody response to the CSP variants was not associated with the presence of the variant in the infection. Significant differences were observed between the single nucleotide polymorphism (SNP) -511T>C in the IL1B gene and levels of antibodies to the VK247 and P. vivax-like variants, but there were no associations between SNPs in IL1 and IL2 genes and their plasma products. CONCLUSIONS: Individuals with the rs16944 CC genotype in the IL1β gene have higher antibody levels to the CSP of P. vivax of VK247 and P. vivax-like variants

Enteroparasite and vivax malaria co-infection on the Brazil-French Guiana border: Epidemiological, haematological and immunological aspects - Fig 3

<p>Serum levels of TNF-α (a), IFN-γ (b), IL-2 (c), IL-4 (d), IL-5 (e) and IL-10 (f) cytokines in pg/mL among the following groups: endemic control, malaria, enteroparasite and co-infected groups. Multiple correlations were made using the non-parametric Kruskal-Wallis test followed by the Dunn's post hoc test. Data are expressed in box plot format (minimum to maximum values, P25%–P75% and median). Significant differences were estimated using the median values for each group, with p < 0.05 being considered significant. * p = 0.05, ** p < 0.05 and *** p = 0.001.</p

Epidemiological and haematological data for the studied groups.

<p>Epidemiological and haematological data for the studied groups.</p

Distribution and number of individuals among groups and subgroups according to the malaria and intestinal parasite diagnosis.

<p>Distribution and number of individuals among groups and subgroups according to the malaria and intestinal parasite diagnosis.</p

Enteroparasite and vivax malaria co-infection on the Brazil-French Guiana border: Epidemiological, haematological and immunological aspects - Fig 1

<p>(a) Frequency-specific antibody response to PvMSP-1₁₉, as determined by ELISA. The subjects were grouped into responders and non-responders to the recombinant protein. (b) Prevalence of anti-PvMSP-₁₉ IgG antibodies in the studied groups. (c) PvMSP-1₁₉ reactivity index (RI) between the studied groups as expressed in box plot format, with individual data shown as points. Multiple correlations were made using the nonparametric Kruskal-Wallis test followed by Dunn’s post hoc test (minimum to maximum values, P25%–P75% and median); significant differences were estimated using the median values for each group, and those with p < 0.05 were considered significant. ** p < 0.05, *** p = 0.001 and **** p < 0.001.</p