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Toxoplasmosis causes serious harm to the fetus, as tachyzoite dissemination, during pregnancy in women developing the primo-infection. The microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. Circulating miRNA are promising biomarkers for diagnosis and prognosis of numerous diseases. The miRNAs levels are estimated by quantitative real-time PCR (qPCR), however, the relative quantification of each miRNA expression requires proper normalization methods using endogenous miRNAs as control. This study analyzed the expression of three endogenous miRNAs (miR-484, miR -423-3p and miR-26b-5p) for use as normalizers in future studies of target miRNAs for gestational toxoplasmosis (GT). A total of 32 plasma samples were used in all assays divided in 21 from women with GT and 11 from healthy women. The stability of each endogenous miRNA was evaluated by the algorithm methods RefFinder that included GeNorm, Normfinder, BestKeeper and comparative delta-CT programs. The miR-484 was the most stably gene, and equivalently expressed in GT and NC groups. These results contribute to future studies of target miRNAs in clinical samples of women with gestational toxoplasmosis.

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Duffy blood group phenotypes [Fy(a + b-), Fy(a-b+), Fy(a + b+), Fy(a-b-)], characterized by the expression of Fya, and Fyb antigens, are present in red blood cells. Therefore, we hypothesize that the non-hematopoietic expression of these antigens might influence cell invasion by T. gondii. 576 consecutive patients from both genders were enrolled. The presumed OT clinical diagnosis was performed. Duffy phenotyping was performed by hemagglutination in gel columns and for the correct molecular characterization Fy(a-b-) phenotype, using PCR-RFLP. Anti-T. gondii IgG antibodies were detected by ELISA. Chi-square, Fisher's exact tests were used to compare the proportions. OT was present in 22.9% (n = 132) and absent in 77.1% (n = 444) of patients. The frequencies of anti-T. gondii IgG antibodies were higher in OT (127/132, 96.2%) than those without this disease (321/444, 72.3%) (p < .0001). None of the Duffy antigens or phenotypes were associated with T. gondii infection (χ2: 2.222, GL: 3, p = .5276) as well as the risk of OT (χ2: 0.771, GL: 3, p = .8566). Duffy blood group system phenotypes and their antigens do not constitute risk factors for infection by T. gondii infection and the development of OT.

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This study investigated the potential of five miRNA candidates for cerebral toxoplasmosis/HIV co-infection (CT/HIV) biomarkers. miR-155-5p, miR-146a-5p, miR-21-5p, miR-125b-5p and miR-29c-3p were tested in 79 plasma divided into groups: 32 CT/HIV patients; 27 individuals with asymptomatic toxoplasmosis (AT); and 20 individuals seronegative for toxoplasmosis (NC). From each was collected peripheral blood/EDTA for laboratory diagnosis. Blood cells for DNA extractions (molecular diagnosis), plasma for RNA extractions (gene expression) and ELISA (serological diagnosis). miRNA expression was performed by qPCR, and values were expressed in Relative Quantification (RQ). Among the five miRNAs, miR-21-5p and miR-146a-5p were up-expressed in CT/HIV group when compared with AT and NC groups. RQ means for miR-21-5p and miR-146a-5p in CT/HIV group were 3.829 and 2.500, while in AT group, were 1.815 and 1.661, respectively. Differences between 3 groups were statistically significant (Kruskal-Wallis ANOVA test), as well as CT/HIV and AT groups (Mann-Whitney test). Plasma of CT/HIV and AT groups expressed similar levels of miR-29c-3p, miR-155-5p and miR-125b-5p. As NC group was different of CT/HIV and AT groups, differences between three groups were statistically significant (Kruskal-Wallis ANOVA test). No difference was shown between CT/HIV and AT groups (Mann-Whitney test). These results suggest the host miRNAs modulation by Toxoplasma gondii.

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Gene expression analyses based on messenger RNA (mRNA) expression require accurate data normalization. When using endogenous reference genes, these should be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context. This study was aimed to identify reference genes that have more stable mRNA levels among individuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brain autopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6 endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP), beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MT-ATP6). Furthermore, validation of their stabilities and performance as reference genes was determined by geNorm and NormFinder programs. The results show that the most stable genes for PBMC and fresh skin biopsies were TBP and UBC; in FFPE lung autopsies and skin biopsies were GAPDH and B2M; and in FFPE brain autopsies were GAPDH and UBC. In addition, 18S rRNA was the least stable of all genes analyzed. These data concluded that even genes constitutively expressed have transcript level variations in different tissues as well as storage and experimental conditions. These observations suggest that suitable reference genes should be selected for normalization of gene expression data analysis.

As análises de expressão gênica baseadas na expressão do RNA mensageiro (mRNA) requerem normalização precisa dos dados. Ao usar genes de referência endógenos, estes devem ser cuidadosamente validados. Os genes de referência validados variam muito, dependendo do tecido, subconjuntos de células e contexto experimental. Este estudo teve como objetivo identificar genes de referência que apresentam níveis de mRNA mais estáveis ​​entre indivíduos em células mononucleares do sangue periférico (PBMC); biópsias de pele fresca; autópsias pulmonares e cerebrais, bem como biópsias de pele fixadas em formalina e embebidas em parafina (FFPE). Portanto, 6 genes de referência endógenos foram avaliados por reação quantitativa em cadeia da polimerase em tempo real: rRNA 18S, gliceraldeído-3-fosfato desidrogenase (GAPDH), proteína de ligação à caixa TATA (TBP), beta-2-microbolina (B2M), ubiquitina C (UBC) e ATP sintase 6 mitocondrialmente codificada (MT-ATP6). Além disso, a validação de suas estabilidades e desempenho como genes de referência foi determinada pelos programas geNorm e NormFinder. Os resultados mostram que os genes mais estáveis ​​para PBMC e biópsias de pele fresca foram TBP e UBC; nas autópsias pulmonares de FFPE e biópsias de pele foram GAPDH e B2M; e nas autópsias cerebrais de FFPE foram GAPDH e UBC. Além disso, o 18S rRNA foi o menos estável de todos os genes analisados. Esses dados concluíram que mesmo os genes expressos constitutivamente apresentam variações no nível de transcrição em diferentes tecidos, bem como condições experimentais e de armazenamento. Essas observações sugerem que genes de referência adequados devem ser selecionados para normalização da análise dos dados de expressão gênica.

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Gene expression analyses based on messenger RNA (mRNA) expression require accurate datanormalization. When using endogenous reference genes, these have to be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context.The aim of this study was to identify reference genes that present more stable mRNA levels amongindividuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brainautopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP),beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MTATP6).Furthermore, validation of their stability and suitability as reference genes was determined bythe geNorm program. The results show that in PBMC and fresh skin biopsies, TBP and UBC wereidentified as the most stable, while in FFPE lung autopsies and skin biopsies, GAPDH and B2M, andin FFPE brain autopsies, GAPDH and UBC turned out to be the most stable...

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