Potential biomarkers for the clinical prognosis of severe dengue

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.

Parasite load and risk factors for poor outcome among children with visceral leishmaniasis. A cohort study in Belo Horizonte, Brazil, 2010-2011

Clinical and laboratory risk factors for death from visceral leishmaniasis (VL) are relatively known, but quantitative real-time polymerase chain reaction (qPCR) might assess the role of parasite load in determining clinical outcome. The aim of this study was to identify risk factors, including parasite load in peripheral blood, for VL poor outcome among children. This prospective cohort study evaluated children aged ≤ 12 years old with VL diagnosis at three times: pre-treatment (T0), during treatment (T1) and post-treatment (T2). Forty-eight patients were included and 16 (33.3%) met the criteria for poor outcome. Age ≤ 12 months [relative risk (RR) 3.51; 95% confidence interval (CI) 1.89-6.52], tachydyspnoea (RR 3.46; 95% CI 2.19-5.47), bacterial infection (RR 3.08; 95% CI 1.27-7.48), liver enlargement (RR 3.00; 95% CI 1.44-6.23) and low serum albumin (RR 7.00; 95% CI 1.80-27.24) were identified as risk factors. qPCR was positive in all patients at T0 and the parasite DNA was undetectable in 76.1% of them at T1 and in 90.7% at T2. There was no statistical association between parasite load at T0 and poor outcome.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Experimental infection with the Toxoplasma gondii ME-49 strain in the Brazilian BR-1 mini pig is a suitable animal model for human toxoplasmosis

Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation with pigs is necessary for the development of new therapeutic approaches to human diseases. BR-1 mini pigs were intramuscularly infected with T. gondii with tachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology and serum biochemistry were analysed and buffy coat cells were inoculated in mice to determine tachyzoite circulation. No alterations were observed in erythrocyte and platelet values; however, band neutrophils increased seven days after infection with ME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulating ME-49 or RH strain tachyzoites in the pigs' peripheral blood at two and seven or nine days post-infection. The tachyzoites were also directly observed in blood smears from the infected pigs outside and inside leucocytes for longer periods. Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90 days, the pigs were euthanised and their tissue samples were processed and inoculated into mice. The mice serology revealed the presence of parasites in the hearts, ileums and mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were only observed after pig heart tissue inoculation. The infected pigs presented similar human outcomes with relatively low pathogenicity and the BR-1 mini pig model infected with ME-49 is suitable to monitor experimental toxoplasmosis.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Enhancement of viability of radiosensitive (PBMC) and resistant (MDA-MB-231) clones in low-dose-rate cobalt-60 radiation therapy

AbstractObjective:In the present study, the authors investigated the in vitrobehavior of radio-resistant breast adenocarcinoma (MDA-MB-231) cells line and radiosensitive peripheral blood mononuclear cells (PBMC), as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy.Materials and Methods:The cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed.Results:Radiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation.Conclusion:Low dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer.

Chromosomal abnormalities in couples with recurrent first trimester abortions

PURPOSE: To investigate the prevalence of chromosomal abnormalities in couples with two or more recurrent first trimester miscarriages of unknown cause. METHODS: The study was conducted on 151 women and 94 partners who had an obstetrical history of two or more consecutive first trimester abortions (1-12 weeks of gestation). The controls were 100 healthy women without a history of pregnancy loss. Chromosomal analysis was performed on peripheral blood lymphocytes cultured for 72 hours, using Trypsin-Giemsa (GTG) banding. In all cases, at least 30 metaphases were analyzed and 2 karyotypes were prepared, using light microscopy. The statistical analysis was performed using the Student t-test for normally distributed data and the Mann-Whitney test for non-parametric data. The Kruskal-Wallis test or Analysis of Variance was used to compare the mean values between three or more groups. The software used was Statistical Package for the Social Sciences (SPSS), version 17.0. RESULTS: The frequency of chromosomal abnormalities in women with recurrent miscarriages was 7.3%, including 4.7% with X-chromosome mosaicism, 2% with reciprocal translocations and 0.6% with Robertsonian translocations. A total of 2.1% of the partners of women with recurrent miscarriages had chromosomal abnormalities, including 1% with X-chromosome mosaicism and 1% with inversions. Among the controls, 1% had mosaicism. CONCLUSION: An association between chromosomal abnormalities and recurrent miscarriage in the first trimester of pregnancy (OR=7.7; 95%CI 1.2--170.5) was observed in the present study. Etiologic identification of genetic factors represents important clinical information for genetic counseling and orientation of the couple about the risk for future pregnancies and decreases the number of investigations needed to elucidate the possible causes of miscarriages.

Polymorphism in the lymphotoxin-alpha gene, position +252 (rs909253), is not associated with preeclampsia development in Brazilian women

PURPOSE: To investigate the frequencies of polymorphic allele and genotypes for the LT-α gene, position +252 (rs909253), in Brazilian women with preeclampsia.METHODS: This is a case-control study, in which 30 women with preeclampsia, classified according to the criteria of the National High Blood Pressure Education Program, and 115 women in the control group, with at least two healthy pregnancies, were selected. Peripheral blood was collected, and DNA was extracted, followed by genotyping, using specific primers and restriction analysis. The genotypes obtained were AA, AG and GG. Statistical analysis was performed using the χ2association test. The Hardy-Weinberg Equilibrium was tested using the Haploview Program.RESULTS: The results showed no association between genotypes and preeclampsia development (χ2=2.0; p=0.4). When the AG and GG genotypes were grouped according to allele G presence or absence (genotype AA), the data showed that the presence of allele G was not significantly different between cases (women with preeclampsia) and controls (χ2=0.0; p=1.0). The LT-α gene polymorphism, position +252 (rs909253), seems not to be an important candidate for the development of preeclampsia. Other inflammatory genes should be researched, and studies involving gene-environment interactions should be performed, in order to reach a better understanding of the etiology of the preeclampsia.

Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5% of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

Babesia canis vogeli infection in dogs and ticks in the semiarid region of Pernambuco, Brazil

Abstract:This study aimed to report the prevalence of Babesia canis vogeli in dogs and ticks in the urban and rural areas of Petrolina, Pernambuco. Serum and peripheral blood samples of 404 dogs were tested by indirect immunofluorescence assay (IFA) and by blood smears, respectively. The presence of tick infestation was evaluated, and some specimens were submitted to DNA amplification by polymerase chain reaction (PCR). The presence of antibodies anti-B. canis vogeli was determinate in 57.9% (234/404) of dogs. The direct detection of Babesia spp was obtained in 0.5% (2/404) dogs by visualization of intraerythrocytic forms. Infestation by Rhipicephalus sanguineus sensu lato was observed in 54.5% (220/404) of dogs in both urban and rural areas. DNA of Babesia canis vogeli were obtained by PCR in 6% individual (3/50) and 8.7% of pool of ticks (7/80). The risk factors for the presence of anti-B. canis vogeli antibodies, as determined through the application of logistic regression models (P<0.05), were the following: medium breed size variables (P<0.001); contact with areas of forest (P=0.021); and access on the street (P=0.046). This study describes, for the first time, the confirmation of infection of B. canis vogeli in dogs and ticks in the semiarid region of Pernambuco, Brazil.

Trisomy 13 mosaicism demonstrated only in skin fibroblasts in a patient presenting psychomotor retardation, pigmentary dysplasia and some dysmorphic features

A Brazilian female infant presented delayed psychomotor development, skin pigmentary dysplasia and some dysmorphic features. Chromosome analysis from peripheral blood culture was normal, but the karyotype from skin fibroblasts revealed mosaicism for trisomy 13. This case demonstrates the relevance of performing chromosomal analysis of skin fibroblasts in patients with mental retardation, associated with pigmentary dysplasia of the skin and a normal karyotype in peripheral blood lymphocytes. To our knowledge, it is the first report of trisomy 13 demonstrated only in skin fibroblasts.