Hélida Monteiro de Andrade

Proteomic analysis of Trypanosoma cruzi resistance to Benznidazole.

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.

Leishmania (Leishmania) chagasi is not vertically transmitted in dogs

The most frequent and most important mode of human or canine visceral leishmaniasis (CVL) transmission is through the bite of infected sand flies. This study investigates Leishmania (Leishmania) chagasi vertical transmission in offspring of naturally infected dogs. Thus 63 puppies from 18 female dogs with CVL were used. Parasite presence was evaluated through parasitologic and histopathologic examination of lymphatic organs, as well as polymerase chain reaction (PCR) on samples from adults (milk, uterus, placenta, spleen, liver and bone marrow) and offspring (spleen, liver, lymph nodes and bone marrow). PCR sensitivity and specificity were calculated using a microscope as the gold standard on samples of bone marrow, spleen and liver. Specificity was 100% for all organs and sensitivity was 100% for bone marrow, 71.4% for spleen and 66.6% for liver. Bone marrow smears (n = 63), histopathology and imprint of spleen (n = 25), liver (n = 25) and lymph nodes (n = 25) were performed to evaluate congenital transmission in the 63 offspring. PCR was done on 92 samples collected from 56 of the offspring. No test performed on the offspring was positive. It was not possible to confirm vertical transmission of CVL (95% confidence interval for the observed prevalence), despite positive PCR in the placenta of seropositive adults.

Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine.

Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines.

Breast cancer proteomics: a review for clinicians

PurposeBreast cancer is one of the major health problems of the Western world. Although the survival rate has improved with progress in screening and adjuvant systemic therapies, one-third of the patients with initial breast tumor have recurrence of the disease 10 years after the diagnosis, demonstrating the presence of micrometastasis. The underlying molecular mechanism of the disease needs to be better understood. Allied to genomics, proteomics technologies promise to be valuable for identifying new markers that improve screening, early diagnosis, prognosis and prediction of therapeutic response or toxicity, as well as the identification of new therapeutic targets. In this review, we present features of proteomic technology and its main implications, focusing on the protein profile in tumor tissues/cells through MALDI/SELDI, as well as on the current proteomic challenges in the breast cancer study.MethodsWe performed a research of protein profiling studies using mass spectrometry in breast cancer to identify potential biomarkers.ResultsMany protein peaks have been reported to bear significant diagnostic, prognostic or predictive value; however, the candidate biomarkers have not been validated for use in clinical patient care.ConclusionsProteomics is under development and, despite technical barriers that precede the use of proteomics analysis in clinical practice and breast cancer complexity, MALDI-TOF/SELDI-TOF MS proteomic platforms with their innovations are powerful analytical tools for the detection of better protein biomarkers, since the studies are conducted with adequate statistical power and analytical rigor. In the near future, they will be able to fulfill their role in personalized medicine.

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species.

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.

Proteomic analysis of Escherichia coli with experimentally induced resistance to piperacillin/tazobactam

The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.

High-Throughput Analysis of Synthetic Peptides for the Immunodiagnosis of Canine Visceral Leishmaniasis

Background Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis. Methodology/Principal Findings Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs. Conclusions/Significance Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil.

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.

Proteomic analysis of human mesenchymal stromal cells derived from adipose tissue undergoing osteoblast differentiation.

BACKGROUND AIMS Stem cells derived from human adipose tissue (ASC) have the capacity for renewal, are easily obtained and have plasticity properties that allow them to differentiate into several cell types, including osteoblast cells. With the aim of understanding the issue of the osteogenic process and finding reliable biomarkers in cells undergoing the osteogeneic differentiation process, this work took advantage of a proteomic approach to identify proteins involved in osteogenesis. METHODS For this purpose, ASC were analyzed under three conditions: S0, in the absence of stimulation; S1, with 2 weeks of osteogenic medium stimulation; and S2, with 4 weeks of osteogenic medium stimulation. The identification of ASC was carried out by flow cytometry using antibodies specific to known undifferentiated stem cell-surface markers. Cell viability, enzymatic activity, mineral deposition, collagen structure and production and gene analyzes were evaluated for each condition. RESULTS Phenotypic modifications were observed during the in vitro osteogenic differentiation process by two-dimensional (2-D) differential image gel electrophoresis (DIGE). The proteins were identified by mass espectrometry in tandem (MS/MS) analyzes using Matrix-assisted laser desorption/ionization with TOF/TOF is a tandem mass spectrometry method where two time-of-flight mass spectrometers are used consecutively (MALDI-TOF/TOF). A total of 51 differentially expressed proteins was identified when comparing the three observed conditions. Sixteen different spots were identified in the S0 stage compared with S2, while 28 different spots were found in S2 compared with S0. S1 expressed seven different spots compared with S0 and S2. CONCLUSIONS These findings suggest the involvement of several proteins directly related to the osteogenic pathway, which can be used to improve understanding of the osteogenic process.

Proteomic analysis of the soluble proteomes of miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates obtained from Brazilian patients with different treatment outcomes

UNLABELLED The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. BIOLOGICAL SIGNIFICANCE Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.