Theranostic applications of phage display to control leishmaniasis: Selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

© 2015, Sociedade Brasileira de Medicina Tropical. All rights reserved. Phage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.Peer Reviewe

Evaluation of peak inspiratory pressure, tidal volume and respiratory rate during ventilation of premature lambs using a self-inflating bag

OBJECTIVE: To evaluate the peak inspiratory pressure, tidal volume and respiratory rate achieved during manual ventilation of premature lambs, using a self-inflating bag. METHODS: In this descriptive, experimental study, five pairs of physicians, selected at random among 35 neonatologists working at a neonatal intensive care unit and with experience in the resuscitation of newborn infants, ventilated five intubated premature lambs using a self-inflating bag. Pressure and flow monitor signals were passed through a transducer and digitized for recording and analysis. Tidal volume and pressure curves were obtained from the integral of flow rate, at peak, during the last 50 seconds of every fifth minute, and analyzed. RESULTS: Median pressure was 39.8 (IQ25-75% 30.2-47.2) cmH2O; being below 20 in 1.1% of cases and above 40 in 49.1%. Seven out of 10 physicians produced more than six pressure peaks of over 40 cmH2O. Median tidal volume/kg was 17.8 (IQ25-75% 14.1-22.4) mL, being below 5 mL in 0.1% of cases and greater than or equal to 20 mL in 37.7%. All of the physicians propelled five or more ventilation cycles with tidal volume/kg of 20 mL or more. Respiratory rate was between 30 and 60 cycles/minute in 65.9% of cases, being below 30 in 6.8% of cases and over 60 in 27.3% of cases. CONCLUSIONS: There was major variation in peak inspiratory pressure and tidal volume/kg values, which were in many cases elevated, attaining levels that habitually cause biotrauma, while respiratory rates were adequate in the majority of cases.OBJETIVO: Avaliar o pico de pressão inspiratória, o volume corrente e a freqüência respiratória obtidos durante ventilação manual de carneiros prematuros, utilizando balão auto-inflável. MÉTODOS: Estudo experimental descritivo em que cinco duplas de médicos selecionados aleatoriamente entre 35 neonatologistas que trabalham em unidade de terapia intensiva neonatal e experientes em reanimação de recém-nascidos ventilaram cinco carneiros prematuros intubados, utilizando balão auto-inflável. Os sinais de pressão e fluxo eram captados, convertidos por meio de transdutores e digitalizados para armazenamento e análise. Foram avaliadas curvas de pressão e de volume corrente, este a partir da integral do fluxo, em suas medidas de pico, nos 50 segundos finais de cada 5 minutos. RESULTADOS: A mediana da pressão foi de 39,8 (IQ25-75% 30,2-47,2) cmH2O; foi menor que 20 em 1,1% das vezes e maior que 40 em 49,1%. Sete em 10 médicos propiciaram mais de seis picos de pressão maiores que 40 cmH2O. A mediana do volume corrente/kg foi de 17,8 (IQ25-75% 14,1-22,4) mL, sendo menor que 5 mL em 0,1% das vezes e igual ou maior que 20 mL em 37,7%. Todos os médicos impuseram cinco ou mais ciclos ventilatórios com volume corrente/kg de 20 mL ou mais. A freqüência situou-se entre 30 e 60 ciclos/minuto em 65,9% das vezes, sendo menor que 30 em 6,8% e maior que 60 em 27,3% das vezes. CONCLUSÃO: Ocorreu grande variabilidade nos valores do pico de pressão inspiratória e do volume corrente/kg, sendo muitas vezes elevados e alcançando níveis indutores de biotrauma; para a freqüência respiratória, os valores foram adequados na maioria das vezes.Universidade de BrasíliaUnBUniversidade Federal de São Paulo (UNIFESP)UnB Hospital Universitário da UnBUniversidade de São Paulo Faculdade de Medicina Instituto da Criança do Hospital das ClínicasUNIFESPSciEL

Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads' draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti-Leishmania IgG2a antibodies and a predominant IL-12-driven IFN-γ production (mainly produced by CD4 T cells) against parasite proteins, whereas unprotected controls showed anti-Leishmania IgG1 antibodies and parasite-mediated IL-4 and IL-10 responses. Vaccinated mice showed an anti-LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD-specific IFN-γ, IL-12 and GM-CSF cytokines before and after infection. The protection was correlated with the Leishmania-specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.Instituto Nacional de Ci^encia e Tecnologia em Nano-biofarmac^eutica (INCT-NanoBiofar), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-482976/2012-8, APQ-488237/2013-0 and APQ-467640/2014-9). In addition, this study was partially funded by the Spanish grant from Ministerio de Economía y Competitividad-FEDER (FIS PI14/00366 from the Instituto de Salud Carlos III)Peer Reviewe

A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis

Background: LiHyV is an antigenic hypothetical protein present in both promastigote and amastigote stages of Leishmania infantum, which was recently identified by an immunoproteomic approach. A recombinant version of this protein (rLiHyV) was evaluated as a diagnostic marker for canine VL (CVL). In addition, the prophylactic efficacy of the rLiHyV protein, and two of its CD8+ T cell epitopes, has been analyzed in a murine model of visceral leishmaniasis (VL). Methods: Initially, the rLiHyV protein was evaluated by an ELISA technique for the serodiagnosis of CVL. Secondly, vaccines composed of the recombinant protein and both chemically synthesized peptides, combined with saponin as an adjuvant; were administered subcutaneously into BALB/c mice. The cellular and humoral responses generated by vaccination were evaluated. In addition, the parasite burden and immune response were studied 10 weeks after L. infantum infection. Results: The rLiHyV protein was recognized by antibodies of VL dogs. No cross-reactivity was obtained with sera from dogs vaccinated with a Brazilian commercial vaccine, with sera from animals infected with Trypanosoma cruzi, Babesia canis and Ehrlichia canis, or those from non-infected animals living in an endemic area for leishmaniasis. After challenge with L. infantum, spleen cells of BALB/c mice vaccinated with rLiHyV/saponin stimulated with parasite antigens showed a higher production of IFN-γ, IL-12 and GM-CSF, than the same cells obtained from mice vaccinated with the individual peptides, or mice from control (inoculated with saline or saponin) groups. This Th1-type cellular response observed in rLiHyV/saponin vaccinated mice was accompanied by the induction of parasite-specific IgG2a isotype antibodies. Animals immunized with rLiHyV/saponin showed significant reductions in the parasite burden in the liver, spleen, bone marrow and in the lymph nodes draining the paws relative to control mice. Conclusions: The present study showed for the first time that the L. infantum LiHyV protein could be considered as a vaccine candidate against L. infantum infection, as well as a diagnostic marker for CVL.This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8, and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and APF are grant recipient of CNPq.Peer Reviewe

Development of photocatalytic 3D-Printed cementitious mortars: influence of the curing, spraying time gaps and TiO2 coating rates

This work evaluated the photocatalytic activity of 3D-printed cementitious mortar specimens functionalized with TiO2 nanoparticles to obtain a multifunctional and smart concrete. This research aims to assess the influence of different parameters related to the functionalization process such as adsorption, coating time gaps, and coating rates on the degradation efficiency of the functionalized cementitious specimens. Each specimen was evaluated under the degradation of Rhodamine B (RhB) in an aqueous solution using a sun-light simulation. The obtained results showed a decrease in adsorption (under dark condition) with increasing the sample curing age. The highest photocatalytic efficiency was observed for coated samples aged 7 days. By increasing the coating rates, the photocatalytic efficiency is enhanced. Nonetheless, regardless of the coating rates, all the specimens showed an increase in photocatalytic efficiency for longer time periods of light exposition, i.e., after 8 h of irradiationThis work was partly financed by FCT/MCTES through national funds (PIDDAC) under the R&D Unit Institute for Sustainability and Innovation in Structural Engineering (ISISE), under reference UIDB/04029/2020. The authors acknowledge the support of the DST group construction company for funding the project Chair dst/IB-S: Smart Systems for Construction. The first two authors would like to acknowledge the PhD grants SFRH/BD/143636/2019 and SFRH/BD/137421/2018 provided by the Portuguese Foundation for Science and Technology (FCT). Additionally, the authors would like to acknowledge FCT for the financing this research work by the project NanoAir PTDC/FIS-MAC/6606/2020 and the Strategic Fundings UIDB/04650/2020-2023 and UIDB/04029/202

Transcriptome and Proteome of Fish-Pathogenic Streptococcus agalactiae Are Modulated by Temperature

Streptococcus agalactiae is one of the most important pathogens associated with streptococcosis outbreaks in Nile tilapia farms worldwide. High water temperature (above 27°C) has been described as a predisposing factor for the disease in fish. At low temperatures (below 25°C), fish mortalities are not usually observed in farms. Temperature variation can modulate the expression of genes and proteins involved in metabolism, adaptation, and bacterial pathogenicity, thus increasing or decreasing the ability to infect the host. This study aimed to evaluate the transcriptome and proteome of a fish-pathogenic S. agalactiae strain SA53 subjected to in vitro growth at different temperatures using a microarray and label-free shotgun LC-HDMSE approach. Biological triplicates of isolates were cultured in BHIT broth at 22 or 32°C for RNA and protein isolation and submitted for transcriptomic and proteomic analyses. In total, 1,730 transcripts were identified in SA53, with 107 genes being differentially expressed between the temperatures evaluated. A higher number of genes related to metabolism, mainly from the phosphotransferase system (PTS) and ATP-binding cassette (ABC) transport system, were upregulated at 32°C. In the proteome analysis, 1,046 proteins were identified in SA53, of which 81 were differentially regulated between 22 and 32°C. Proteins involved in defense mechanisms, lipid transport and metabolism, and nucleotide transport and metabolism were upregulated at 32°C. A higher number of interactions were observed in proteins involved in nucleotide transport and metabolism. We observed a low correlation between the transcriptome and proteome datasets. Our study indicates that the transcriptome and proteome of a fish-adapted S. agalactiae strain are modulated by temperature, particularly showing differential expression of genes/proteins involved in metabolism, virulence factors, and adaptation

Evaluation of peak inspiratory pressure and respiratory rate during ventilation of an infant lung model with a self-inflating bag

OBJETIVO: Avaliar o pico de pressão inspiratória e a freqüência ventilatória obtidos por médicos quando da utilização de balão auto-inflável neonatal em modelo de pulmão. MÉTODOS: Quinze médicos ventilaram simuladores de pulmão de recém-nascidos a termo e pré-termo, sendo os desfechos captados através de um monitor de ventilação. RESULTADOS: As medianas dos picos de pressão foram 23 (intervalo interquartil, 15-47) e 26 (intervalo interquartil, 14-51) cmH2O, sendo menor que 20 em 41,2 e 35,8% das vezes; maior que 40 em 29,7 e 33,6% das vezes e entre 27 e 33 cmH2O em 8,2 e 6,5% das vezes, para o pulmão de termo e de pré-termo, respectivamente. As medianas das freqüências ventilatórias foram de 45 (intervalo interquartil, 36-57) e 48 (intervalo interquartil, 39-55,5) ciclos por minuto, sendo menor que 30 em 9,3 e 6,7% das vezes e maior que 60 em 12 e 13,3% das vezes, respectivamente, para o pulmão de termo e de pré-termo. As diferenças nas medianas não foram estatisticamente significantes. CONCLUSÃO: A ventilação utilizando o balão auto-inflável promoveu adequação para a freqüência ventilatória em aproximadamente 80% das vezes, e os médicos não conseguiram promover ventilação com mínima variabilidade na pressão, alcançando níveis diferentes daqueles definidos no curso de reanimação neonatal em 70% das vezes. Essa situação independeu da ventilação do pulmão-teste análogo ao sistema respiratório de recém-nascidos a termo ou pré-termo.OBJECTIVE: To evaluate the peak inspiratory pressure and ventilation rate achieved by physicians when using a neonatal self-inflating bag on a lung model. METHODS: Fifteen physicians ventilated full term and preterm infant lung simulators while the outcomes were captured by a ventilation monitor. RESULTS: Median peak pressures in cmH2O for full term and preterm lungs were 23 (interquartile range: 15-47) and 26 (interquartile range: 14-51), being less than 20 in 41.2 and 35.8% of the pressure curves analyzed, more than 40 in 29.7 and 33.6%, and between 27 and 33 cmH2O in 8.2 and 6.5% of the curves, respectively. Median ventilation rates were 45 (interquartile range: 36-57) and 48 (interquartile range: 39-55.5) cycles per minute, being more than 30 in 9.3 and 6.7% of pressure curves and more than 60 in 12 and 13.3% of pressure curves, for the full term and preterm lungs, respectively. The differences between these medians were not statistically significant. CONCLUSIONS: Ventilation rates achieved with the self-inflating bag were adequate in approximately 80% of pressure curves analyzed, but the physicians were unable to provide ventilation with minimal pressure variation, producing pressures that diverged from those defined by the neonatal resuscitation training course in 70% of the curves. This was irrespective of whether they were ventilating the lung model analogous to preterm or full term infant lungs

Out-of-focus Blur: Image De-blurring

Image de-blurring is important in many cases of imaging a real scene or object by a camera. This project focuses on de-blurring an image distorted by an out-of-focus blur through a simulation study. A pseudo-inverse filter is first explored but it fails because of severe noise amplification. Then Tikhonov regularization methods are employed, which produce greatly improved results compared to the pseudo-inverse filter. In Tikhonov regularization, the choice of the regularization parameter plays a critical rule in obtaining a high-quality image, and the regularized solutions possess a semi-convergence property. The best result, with the relative restoration error of 8.49%, is achieved when the prescribed discrepancy principle is used to decide an optimal value. Furthermore, an iterative method, Conjugated Gradient, is employed for image de-blurring, which is fast in computation and leads to an even better result with the relative restoration error of 8.22%. The number of iteration in CG acts as a regularization parameter, and the iterates have a semi-convergence property as well.Comment: 11 page