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Walison Nunes da Silva,1 Pedro Augusto Carvalho Costa,1 Sérgio Ricardo Aluotto Scalzo Júnior,1 Heloísa AS Ferreira,1 Pedro Henrique Dias Moura Prazeres,2 Caroline Leonel Vasconcelos Campos,3 Marco Túllio Rodrigues Alves,1 Natália Jordana Alves da Silva,1 Ana Luiza de Castro Santos,1 Lays Cordeiro Guimarães,1 Maria Eduarda Chen Ferris,1 Ajay Thatte,4 Alex Hamilton,4 Kelly Alves Bicalho,5 Anderson Oliveira Lobo,6 Helton da Costa Santiago,3 Lucíola da Silva Barcelos,1 Maria Marta Figueiredo,7 Mauro Martins Teixeira,3 Vivian Vasconcelos Costa,8 Michael J Mitchell,4 Frédéric Frézard,1 Pedro Pires Goulart Guimaraes1 1Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil; 3Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil; 4Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; 5Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil; 6Department of Materials Engineering, Federal University of Piauí, Teresina, PI, Brazil; 7State University of Minas Gerais, Divinopolis, MG, Brazil; 8Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, MG, BrazilCorrespondence: Pedro Pires Goulart Guimaraes, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, Email ppiresgo@reitoria.ufmg.brIntroduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention.Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1– 7 (Ang(1– 7)) to reduce vascular compression and deposition of extracellular matrix in mice.Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1– 7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization.Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.Keywords: immunotherapy, TRAIL, mRNA, lipid nanoparticle, losartan, angiotensin (1– 7)

Objective: Childhood standing height has been estimated from arm span-related (height AS ) models. The authors aimed to develop and cross-validate a height AS model in individuals with spina bifida (SB) and examine the accuracy of existing height AS models., Methods: Participants were individuals with sacral and low-lumbar SB (n = 14) and non-SB (n = 83), 7-16 years old. Arm span, age, sex, and group (SB vs. non-SB) were candidate height predictors. Sequential regression and leave-one-out cross-validation approaches were used for the model development (M1) and cross-validation (M1-M5). Existing models were: an SB-specific model from Polfuss et al. (M2) and non-SB specific models from Gauld et al. (M3), Mulu et al. (M4), and Zverev et al. (M5) studies., Results: Arm span and group explained 95 % of the variance in height (R 2 = 0.95; p < 0.001; SEE = 3.666 cm) and were included in the M1. Mean differences between actual and estimated height were 0.0 cm (M1), 0.4 cm (M2), and 0.5 cm (M5), all not significant (p > 0.05). However, Bland-Altman analysis revealed some variability in the predictability of the models across participants with limits of agreement ranging from 7.4 to 10.9 cm. Considerable errors were observed with M3 (mean diff: -5.58 cm, 95 % CI: -1.6, -20.2 cm), and M4 (mean diff: 10.5 cm, 95 % CI: -13.8, -27.3 cm)., Conclusions: Models (M1, M2 and M5) may accurately estimate standing height in groups of children with SB. However, due to the wide limits of agreement, caution is recommended when applying these models for individual height estimations., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Editora Ltda.)

To effectively reduce the health impact of coronavirus disease (COVID-19), it is essential to adopt comprehensive strategies to protect individuals from severe acute respiratory syndrome. In that sense, much effort has been devoted to the discovery and repurposing of effective antiviral and anti-inflammatory molecules. The endogenous peptide angiotensin-(1-7) [Ang-(1-7)] has been recently proposed as a promising anti-inflammatory agent to control respiratory infections. Liposomes also emerged as a safe and effective drug carrier system for local drug delivery to the lungs. In this context, the aim of this study was to develop a liposomal formulation of Ang-(1-7) [LAng (1-7)] and investigate its impact on animal survival as well as its antiviral and anti-inflammatory efficacies after intranasal administration in transgenic K18-hACE2 mice infected with SARS-CoV-2. The liposomal formulation was prepared by the ethanol injection method, exhibiting a mean diameter of 100 nm and a polydispersity index of 0.1. Following treatment of infected mice every 12 hours for 5 days, LAng (1-7) extended animal survival compared to the control groups that received either empty liposomes, free Ang-(1-7), or phosphate-buffered saline. Furthermore, the treatment with LAng (1-7) significantly decreased the viral load, as well as IL-6 and tumor necrosis factor levels in the lungs. Conventional treatment with remdesivir by parenteral route used as a positive control promoted similar effects, leading to improved survival rates and reduced viral load in the lungs without significant effects on IL-6 level. In conclusion, liposomal Ang-(1-7) emerges as a promising formulation to improve the treatment and decrease the severity of respiratory infections, such as COVID-19.

Purpose: To perform a cross-cultural adaptation of the OMNI verbal descriptors to Brazilian-Portuguese and examine the validity of the Brazilian version for arm-crank activity., Materials and Methods: Cross-cultural adaptation stages were: permission, translation, synthesis, back translation, expert committee review, pretesting, and submission and appraisal. For the concurrent validity, a Brazilian OMNI-Wheel scale was used to obtain rating of perceived exertion for the overall body (RPE Overall ) and arms (RPE Arms ) in participants ( n  = 9, 10-17 years) with spina bifida. Cardiopulmonary exercise test was used to measure heart rate (HR) and oxygen uptake (VO 2 ). Repeated Measures Correlation ( r rm ) was used to examine the scale validity., Results: The cross-cultural adaptation produced equivalence between English and Brazilian-Portuguese verbal descriptors based on successful translation and pretesting. The Brazilian OMNI-wheel was validated based on strong associations of RPE Overall with VO 2 ( r rm (35) = 0.86, 95% CI [0.93, 0.73], p  < 0.001) and HR ( r rm (35) = 0.89, 95% CI [0.94, 0.79], p  < 0.001) and RPE Arms with VO 2 ( r rm (33) = 0.82, 95% CI [0.91, 0.66], p  < 0.001) and HR ( r rm (33) = 0.82, 95% CI [0.91, 0.66], p  < 0.001)., Conclusions: The OMNI scale was cross-culturally adapted to Brazilian-Portuguese. The Brazilian OMNI-Wheel was validated based on strong associations of RPE with HR and VO 2 . Implications For RehabilitationThe original English OMNI was cross-culturally adapted to Brazilian-Portuguese.A Brazilian wheelchair OMNI was concurrently validated for Arm-Crank Activity in adolescents with spina bifida.This OMNI scale version may aid health providers in monitoring perceived exertion in Brazil.

Introduction: Propulsive wheelchair capacity may be influenced by arm muscle performance. Objectives: To determine whether children and adolescent wheelchair-users with spina bifida show reduced arm muscle strength compared to their typically developing peers and to compare muscle strength data obtained by isokinetic and handheld dynamometry (HHD)., Methods: Participants (mean age 12.5 ± 2.5 years) were assigned to spina bifida (SB; n = 11) or control (n = 22) groups. Isokinetic dynamometry was used to measure peak torque (PT) during dynamic (60°s -1 and 120°s -1 ) and isometric strength testing (MVIC) for shoulder and elbow flexors and extensors. HHD was used to measure MVIC of the same muscle groups., Results: SB showed reduced isokinetic PT for shoulder extensors at 60°s -1 and 120°s -1 , shoulder flexor MVIC, and elbow flexors at 60°s -1 and MVIC; higher PT for shoulder flexors and elbow flexors at 120°s -1 ; and decreased MVIC for elbow flexors but not extensors compared to controls when assessed by HHD. The SB and control groups showed strong positive correlations between MVIC data obtained by the two devices for all muscle groups (r ≥ 0.81; p < .01), except for shoulder flexor MVIC in controls (r = 0.68; p < .01)., Conclusion: SB presented reduced levels of dynamic slow-speed and isometric shoulder and elbow muscle strength and greater dynamic high-speed shoulder and elbow flexor strength than controls possibly due to the exposure to self-sustained wheelchair ambulation. Exercise-based intervention protocols to increase slow-speed arm muscle strength should be considered in youth with SB. Strong positive correlations observed between muscle strength assessed by isokinetic dynamometry and HHD support the use of HHD in this clinical population.

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs., (© 2024. The Author(s).)

CXCL12 is a key chemokine implicated in neuroinflammation, particularly during Zika virus (ZIKV) infection. Specifically, CXCL12 is upregulated in circulating cells of ZIKV infected patients. Here, we developed a lipid nanoparticle (LNP) to deliver siRNA in vivo to assess the impact of CXCL12 silencing in the context of ZIKV infection. The biodistribution of the LNP was assessed in vivo after intravenous injection using fluorescently tagged siRNA. Next, we investigated the ability of the developed LNP to silence CXCL12 in vivo and assessed the resulting effects in a murine model of ZIKV infection. The LNP encapsulating siRNA significantly inhibited CXCL12 levels in the spleen and induced microglial activation in the brain during ZIKV infection. This activation was evidenced by the enhanced expression of iNOS, TNF-α, and CD206 within microglial cells. Moreover, T cell subsets exhibited reduced secretion of IFN-ɣ and IL-17 following LNP treatment. Despite no observable alteration in viral load, CXCL12 silencing led to a significant reduction in type-I interferon production compared to both ZIKV-infected and uninfected groups. Furthermore, we found grip strength deficits in the group treated with siRNA-LNP compared to the other groups. Our data suggest a correlation between the upregulated pro-inflammatory cytokines and the observed decrease in strength. Collectively, our results provide evidence that CXCL12 silencing exerts a regulatory influence on the immune response in the brain during ZIKV infection. In addition, the modulation of T-cell activation following CXCL12 silencing provides valuable insights into potential protective mechanisms against ZIKV, offering novel perspectives for combating this infection., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Masson SAS.)

Background: Plasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines., Methods: A convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax -infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors., Results: Here we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax -infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A 2a receptor., Conclusion: Monocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Diniz, Figueiredo, Costa, Martins-Filho, Teixeira-Carvalho, Pereira, Shugiro Tada, Crocco Afonso, Kohlhoff, Zani, Gazzinelli, Oliveira and Antonelli.)

The current COVID-19 vaccines protect against severe disease, but are not effective in controlling replication of the Variants of Concern (VOCs). Here, we used the existing pre-clinical models of severe and moderate COVID-19 to evaluate the efficacy of a Spike-based DNA vaccine (pCTV-WS) for protection against different VOCs. Immunization of transgenic (K18-hACE2) mice and hamsters induced significant levels of neutralizing antibodies (nAbs) to Wuhan and Delta isolates, but not to the Gamma and Omicron variants. Nevertheless, the pCTV-WS vaccine offered significant protection to all VOCs. Consistently, protection against lung pathology and viral load to Wuhan or Delta was mediated by nAbs, whereas in the absence of nAbs, T cells controlled viral replication, disease and lethality in mice infected with either the Gamma or Omicron variants. Hence, considering the conserved nature of CD4 and CD8 T cell epitopes, we corroborate the hypothesis that induction of effector T-cells should be a main goal for new vaccines against the emergent SARS-CoV-2 VOCs., (© 2023. The Author(s).)

Kupffer cells (KCs) are self-maintained tissue-resident macrophages that line liver sinusoids and play an important role on host defense. It has been demonstrated that upon infection or intense liver inflammation, KCs might be severely depleted and replaced by immature monocytic cells; however, the mechanisms of cell death and the alterations on liver immunity against infections deserves further investigation. We explored the impact of acute Plasmodium infection on KC biology and on the hepatic immune response against secondary infections. Similar to patients, infection with Plasmodium chabaudi induced acute liver damage as determined by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. This was associated with accumulation of hemozoin, increased of proinflammatory response and impaired bacterial and viral clearance, which led to pathogen spread to other organs. In line with this, mice infected with Plasmodium had enhanced mortality during secondary infections, which was associated with increased production of mitochondrial superoxide, lipid peroxidation and increased free iron within KCs-hallmarks of cell death by ferroptosis. Therefore, we revealed that accumulation of iron with KCs, triggered by uptake of circulating hemozoin, is a novel mechanism of macrophage depletion and liver inflammation during malaria, providing novel insights on host susceptibility to secondary infections. Malaria can cause severe liver damage, along with depletion of liver macrophages, which can predispose individuals to secondary infections and enhance the chances of death., (© 2022. The Author(s).)

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli . Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability., Competing Interests: The authors declare no conflict of interest. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2022 The Author(s). Published by Elsevier Ltd.)

We aimed to analyse cardiac autonomic control by assessing the post-exercise heart rate recovery (HRR) and physical fitness in children and adolescents with spina bifida (SB), compared to participants with typical development. A total of 124 participants, 42 with spina bifida (SB group) and 82 typical developmental controls (CO group) performed the arm cranking exercise test with a gas analysis system. HRR was determined at the first (HRR&#95;1) and second (HRR&#95;2) minute at recovery phase. Data are shown as [mean difference (95% CI)]. The SB group showed reduced HR reserve [14.5 (7.1-22.0) bmp, P<0.01], slower HRR&#95;1 [12.4 (7.4-17.5) bpm, P<0.01] and HRR&#95;2 [16.3 (10.6-21.9) bpm; P<0.01], lower VO 2peak [VO 2peak relative: 7.3 (4.2-10.3) mL·min -1 ·kg -1 , P<0.01; VO 2peak absolute: 0.42 (0.30-0.54) L·min -1 , P<0.01], and lower O 2 pulse [2.5 (1.8-3.2) mL·bpm, P<0.01] and ventilatory responses [13.5 (8.8-18.1) L·min -1 , P<0.01] than the CO group. VE/VO 2 was not different between groups [-2.82 (-5.77- -0.12); P=0.06], but the VE/VCO 2 [-2.59 (-4.40-0.78); P<0.01] and the values of the anaerobic threshold corrected by body mass [-3.2 (-5.8- -0.6) mL·min -1 ·kg -1 , P=0.01] were higher in the SB group than in the CO group. We concluded that children and adolescents with SB have reduced physical fitness and a slower HRR response after maximal effort., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria., Competing Interests: The authors have declared that no competing interests exist.

Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

Accurate testing to detect SARS-CoV-2 RNA is key to counteract the virus spread. Nonetheless, the number of diagnostic laboratories able to perform qPCR tests is limited, particularly in developing countries. We describe the use of a virus-inactivating, denaturing solution (DS) to decrease virus infectivity in clinical specimens without affecting RNA integrity. Swab samples were collected from infected patients and from laboratory personnel using a commercially available viral transport solution and the in-house DS. Samples were tested by RT-qPCR, and exposure to infective viruses was also accessed by ELISA. The DS used did not interfere with viral genome detection and was able to maintain RNA integrity for up to 16 days at room temperature. Furthermore, virus loaded onto DS were inactivated, as attested by attempts to grow SARS-CoV-2 in cell monolayers after DS desalt filtration to remove toxic residues. The DS described here provides a strategy to maintain diagnostic accuracy and protects diagnostic laboratory personnel from accidental infection, as it has helped to protect our lab crew.

Methods: A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum -infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum -infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings . Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91)., Conclusion: DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats., Competing Interests: We declare that this work has not been influenced by any financial, personal, or professional interest., (Copyright © 2021 Maria Marta Figueiredo et al.)

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)-a prokaryotic system unable to produce post-translational modifications-or in HEK-293T mammalian cells-a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

Multifunctional interleukin 10 (IL10) + Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4 + and CD8 + T-cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4 + and CD8 + T-cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T-cells like NS3-specific IFNγ/IL10-producing CD4 + T-cells in critical phase and NS3- and ENV-specific CD8 + T-cells producing IFNγ/IL10. In addition, NS3-specific CD8 + T-cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T-cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T-cells. Importantly, multifunctional CD4 + and CD8 + T-cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10 + Th1 and IL10 + Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection., (© 2020 John Wiley & Sons Ltd.)

We describe here the development of an in-house enzyme linked immunosorbent assay (ELISA) for the diagnostic of Chikungunya virus (CHIKV) infections using a recombinant protein from CHIKV. The recombinant protein gene was designed based on 154 sequences and we used computational methods to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKVp) was synthetized and expressed in prokaryotic system. Subsequently, the protein was purified by affinity chromatography and used as antigen in an indirect ELISA. We present data regarding the optimization of the recombinant antigen production and preparation of the ELISA to detect IgG against CHIKV in human sera.

Background: Chikungunya virus (CHIKV) causes a disease characterized by acute onset of fever accompanied by arthralgia. Clinical similarities and co-circulation of other arboviruses such as Dengue virus (DENV) and Zika virus (ZIKV), have complicated their differentiation, making their diagnoses a challenge for the health authorities. Misdiagnosis is a serious issue to the management of patients and development of public health measures., Objectives: We carried out further screening of CHIKV, DENV and ZIKV cases in Minas Gerais, Brazil, after diagnostics were already issued by a state laboratory and according to the Brazilian Ministry of Health (BMH) policy. Our aim was to look for possible co-infections or previous arboviruses' exposure., Study Design: Sera from 193 patients with symptoms of arboviral infections were tested for DEV, ZKV and/or CHIKV by the State laboratory, according to clinical suspicion and following standard BMH guidelines. After an official diagnosis was issued for each patient, we retested samples applying a broader panel of ELISA-based serological tests., Results: We identified 13 patients with concurrent or consecutive infections (IgM positive for more than one arbovirus), including 11 individuals that were positive for CHIKV and other previously confirmed arbovirus infection., Discussion: Guidelines established in many arbovirus-endemic countries prioritizes the diagnosis of Zika and Dengue and no further analyzes are done when samples are positive for those viruses. As a result, possible cases of co-infections with chikungunya are neglected, which affects the epidemiological assessments of virus circulation, patient management, and the development of public health policies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Malaria-associated acute respiratory distress syndrome (MA-ARDS) and acute lung injury (ALI) are complications that cause lung damage and often leads to death. The MA-ARDS/ALI is associated with a Type 1 inflammatory response mediated by T lymphocytes and IFN-γ. Here, we used the Plasmodium berghei NK65 (PbN)-induced MA-ALI/ARDS model that resembles human disease and confirmed that lung CD4 + and CD8 + T cells predominantly expressed Tbet and IFN-γ. Surprisingly, we found that development of MA-ALI/ARDS was dependent on functional CCR4, known to mediate the recruitment of Th2 lymphocytes and regulatory T cells. However, in this Type 1 inflammation-ARDS model, CCR4 was not involved in the recruitment of T lymphocytes, but was required for the emergence of TNF-α/iNOS producing dendritic cells (Tip-DCs) in the lungs. In contrast, recruitment of Tip-DCs and development of MA-ALI/ARDS were not altered in CCR2 -/ - mice. Importantly, we showed that NOS2 -/ - mice are resistant to PbN-induced lung damage, indicating that reactive nitrogen species produced by Tip-DCs play an essential role in inducing MA-ARDS/ALI. Lastly, our experiments suggest that production of IFN-γ primarily by CD8 + T cells is required for inducing Tip-DCs differentiation in the lungs and the development of MA-ALI/ARDS model.

Background: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy., Objective: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL)., Methods: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection., Findings: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis., Main Conclusions: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Myelomeningocele (MMC) is a neural tube defect that often causes spinal cord injury at the thoracolumbar region, as well as sensory and motor paralysis in the lower limbs. This leads to continuous use of a wheelchair and, consequently, a sedentary lifestyle, predisposition to muscle weakness, cardiovascular and respiratory disorders, obesity, and structural alterations in the spine. We assessed the respiratory function and shoulder strength of MMC participants who were wheelchair-users and had no respiratory complaints and compared them to healthy children and adolescents. MMC (n=10) and healthy (n=25) participants of both genders with a mean age of 12.45 years (SD=2.1) were assessed for weight, height, respiratory performance, and isometric peak for shoulder flexors, extensors, abductors, and adductors, using an isokinetic dynamometer. Medullary lesion, functional levels, and abnormal curvatures of the spine were assessed for MMC participants. The level of spinal cord injury for the majority of the MMC participants was high lumbar and they had scoliosis. MMC showed lower values for forced vital capacity, forced expiratory volume at the first second, forced expiratory flow (25-75%), maximal voluntary ventilation, and isometric peak for shoulder flexors and adductors compared to healthy participants. This indicated a decreased vital capacity, respiratory muscle endurance, and shoulder muscle strength.

Background: Cardiovascular autonomic dysfunction is associated with the development of cardiovascular diseases, but little is known about it in children and adolescents with myelomeningocele., Objective: This study investigated the cardiovascular autonomic function in wheelchair-using children and adolescents with myelomeningocele., Methods: Twenty-two participants were assigned to one of two groups: myelomeningocele group (n=11) and Control group (n=11). Heart rate variability and systolic blood pressure variability were collected in supine resting position using spectral analyses. Spontaneous baroreflex sensitivity was collected by time-domain through the sequence method., Results: At rest, heart rate was higher in myelomeningocele group when compared to Control group (mean difference 22.1, 95% CI 4.82-39.40; p=0.01). The heart rate and systolic blood pressure variability parameters did not show differences between groups. However, myelomeningocele showed lower gain mean in baroreflex sensitivity (mean difference -4.5, 95% CI -8.47 to -0.60; p=0.02), when compared to Control., Conclusion: Wheelchair-using children and adolescents with myelomeningocele presented differences in the autonomic cardiovascular function. This may be associated with hypomobility due to wheelchair dependence, and venous muscle pump insufficiency due to paraplegia., (Copyright © 2018 Associação Brasileira de Pesquisa e Pós-Graduação em Fisioterapia. Publicado por Elsevier Editora Ltda. All rights reserved.)

Objective: Isometric muscle strength measured by Handheld dynamometer indicates physical ability. There is no normative data for the Brazilian population. This study aims (a) to describe the development of isometric muscle strength in healthy children and adolescents 5-15 years of age; (b) to evaluate Handheld dynamometer inter and intra-rater reliability., Methods: Isometric muscle strength was obtained for shoulder abduction, elbow and knee flexion and extension, dorsiflexion and plantar flexion in 55 boys and 55 girls, aged between 5 and 15 years. Inter-rater reliability was determined based on the evaluation of 2 raters, with a 20-min interval between them. Intra-rater reliability was based on 2 evaluations from the same rater, one week apart. Interclass correlation coefficient (ICC 2,1; 3,1 ), Bland Altman plots and linear regression models with mixed effects were used to quantify inter and intra-rater reliability, agreement and associations with physical activity level and maturational factors., Results: A linear development of isometric muscular strength was observed for ages between 5 and 10. After age 10, boys showed a larger isometric muscular strength, when compared to girls. Both inter and intra-rater measurements of the Handheld dynamometer are reliable (ICC>0.63)., Conclusions: This study shows increase in isometric muscle strength starting at 10 years of age for boys, when compared to girls and inter and intra-rater reliability for the assessment of isometric strength, using the Handheld dynamometer for the muscle groups tested on the dominant and non-dominant side, for children between 5 and 15 years of age., (Copyright © 2018 Associação Brasileira de Pesquisa e Pós-Graduação em Fisioterapia. Publicado por Elsevier Editora Ltda. All rights reserved.)

The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4+ forkhead box P3 (Foxp3+) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1+Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor+ and interferon-gamma+ cells than PD-1-Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.

Background: Previous studies discussed the repeatability and variability in plantar pressure measurement, but a few considered different age groups. Here we determine within and between-days repeatability and variability of plantar pressure measurement during gait in participants from different age groups., Method: Plantar pressure was recorded in children, young adults and older adults walking at preferred speed in four non-consecutive days within one week. Data from 10 steps from each foot in each day were analyzed considering the different regions of the foot. Mean and peak plantar pressure and data variability were compared between the steps, foot regions and days., Results: To describe mean and peak pressure during gait in children and adults a single measurement can be enough, but elderly will requires more attention especially concerning peak values. Variability in mean pressure did not differ between age groups, but peak pressure variability differed across foot regions and age groups., Conclusion: One single observation can be used to describe plantar pressure during gait in children and adults. When the interest concerns older people, it might be pertinent to consider more than one day of assessment, especially when looking at peak pressure.

Purpose: We analyzed the evolution and pattern of heart rate (HR) during the 12-minute wheelchair propulsion field test (WPFT) and compared the peak HR (HR peak ) from the WPFT to the HR peak obtained in the progressive cardiopulmonary exercise test on arm cranking ergometer (ACT). We aimed to determine if the field test detects the HR peak consistently and could be used in clinical practice., Methods: Eleven wheelchair-using children and adolescents with myelomeningocele (aged 8-15 y) performed a maximal ACT and a 12-minute WPFT. HR was recorded continuously at rest, during each minute of the tests, and at recovery. Mixed analysis of variance was used to compare the variables at rest and peak. Bland-Altman plot and Lin's concordance correlation coefficient were used to show agreement between the tests., Results: During minute 2 of the WPFT, participants reached 73%-96% of the HR peak values recorded in the ACT. From minutes 4 to 12, participants reached HR peak values ranging 86%-109% of the values recorded in the ACT. There is agreement between the ACT and the WPFT tests., Conclusion: WPFT with minimal duration of 4 minutes may be an alternative tool to obtain HR peak in children and adolescents with myelomeningocele.

Background: Fluid-attenuated inversion recovery (FLAIR) vascular hyperintensities (FVHs) are common in patients with acute ischemic stroke, possibly representing impaired hemodynamics in the ischemic territory due to intracranial steno-occlusive disease. There are few reports on FVHs in patients with transient ischemic attack (TIA)., Aims: We investigated the prevalence of FVHs and its clinical correlations in patients with TIA., Methods: We evaluated consecutive patients admitted with TIA from February 2009 to June 2012 who had undergone magnetic resonance imaging within 30 hours of symptoms onset and intracranial and extracranial vascular imaging. Two independent neuroradiologists determined the presence of FVHs. We assessed the relationship between FVHs, clinical presentation, vascular risk factors, neuroimaging characteristics, and the presence of large artery stenosis or occlusion., Results: Seventy-two patients with TIA were evaluated. FVHs were present in 12 (16.7%) patients. The overall agreement between examiners was good (κ = .67). There were no differences in the frequency of intracranial or cervical arterial stenosis in patients with and without FVH. In a multivariate logistic regression analysis including atrial fibrillation (AF), congestive heart failure, and diabetes, only AF remained in the final model., Conclusions: FVH signals on FLAIR images occur in patients with TIA and might correlate with clinical variables like AF and not only with large vessel occlusion. The presence of FVH in patients with TIA and AF might be a surrogate marker for a large vessel occlusion spontaneously recanalized or for impaired autoregulation in a previously ischemic vascular territory., (Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans.

This study aims to provide important preliminary in vivo data on the general biocompatibility and degradation of in situ forming chitosan hydrogels which differ in the type of cross-linking: physical cross-linking (with glycerol phosphate disodium salt) and physico/chemical co-cross-linking (with glycerol phosphate disodium salt and genipin). Additionally, in vitro degradation studies, using lysozyme, were conducted for comparison. The subcutaneous implantation of the liquid formulations of both hydrogels in animal model (Wistar rats) was performed to assess tissue response at the end of two time periods (8 and 30days). Although the properties of both hydrogels are different (namely, pore size and mechanical strength), leading to remarkably distinct in vitro degradation behaviors, the in vivo degradation is quite similar for both types. Regarding biocompatibility, the in vivo evaluation performed by histological analysis, showed a mild to moderate chronic inflammatory reaction with no foreign body reaction, demonstrating that both hydrogels show good results for in vivo biodegradation and biocompatibility, therefore are most adequate for biomedical applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1β) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1β production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1., Methods: C57BL/6 (wild type, WT) and TLR2/9 -/- mice were infected by the intranasal route with HSV-1 (1 × 10 6 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1β, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1β and granzyme B were quantified by real time PCR., Results: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1β and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8 + T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9 -/- infected mice., Conclusion: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8 + T lymphocytes through IL-1β, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

Cardiovascular disease is the leading cause of morbidity and mortality among industrialized countries. Vascular grafts are often required for the surgical treatments. Considering the limitations associated with the use of autografts and with the currently available synthetic materials, a growing demand in tissue engineered vascular grafts has been registered. During the work here described, electrospinning technique was used to prepared fibrous matrices to be applied as vascular implants. For that purpose, electrospun polycaprolactone (PCL) fibrous mats were produced and afterwards coated with different hydrogel formulations based in photocrosslinkable gelatin (GelMA) and the macromers poly(ethylene glycol) acrylate (PEGA) and poly(ethylene glycol) diacrylate (PEGDA). These were further photocrosslinked under UV irradiation using Irgacure ® 2959 (by BASF) as the photoinitiator. The suitability of the coated scaffolds for the intended application, was evaluated by assessing their chemical/physical properties as well as their interaction with blood and endothelial cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Rotating shiftwork is common for air traffic controllers and usually causes sleep deprivation, biological adaptations, and life changes for these workers. This study assessed quality of life, the sleep, and the health of 30 air traffic controllers employed at an international airport in Brazil. The objective was to identify health and quality of life concerns of these professionals. The results identified physical inactivity, overweight, excess body fat, low scores for physical and social relationships, and sleep deprivation for workers in all four workshifts. In conclusion, these workers are at risk for chronic non-transmittable diseases and compromised work performance, suggesting the need for more rest time before working nightshifts and work environments that stimulate physical activity and healthy diets., (© 2016 The Author(s).)

Background: Orthosis use prevents muscle contracture and prolongs ambulation in patients with Duchenne muscular dystrophy (DMD). However, its biomechanical effects on gait are unclear. This study assessed the effects of daytime and night-time use of Articulated Ankle Foot Orthosis (AFO) on kinematic, kinetic and spatial/temporal gait parameters of DMD patients., Methods: Twenty ambulatory patients (4-12years of age) were assigned to one of three groups: no orthosis (NoO; n=7), night-time orthosis (NiO; n=7), day-time orthosis (DO; n=6). All subjects were evaluated once (Ev1) and five of them were re-evaluated between five and seven months after Ev1 (Ev2)., Findings: Cross-sectional analysis with linear mixed-effects models (ANOVA) showed increased peak dorsiflexion angle and dorsiflexor moment and decreased plantar flexion angle and ankle joint power generation for the DOwith group when compared to the NoO group (P<0.05). The DOwith group also showed decreased peak hip flexion angle, hip power absorption, plantar flexion angle and increased peak dorsiflexion moment when compared to the NoO group (P<0.05). Analysis of gait cycle curves showed significant and clinically relevant changes in kinematic and kinetic parameters for the DOwith group when compared to the other experimental groups. Longitudinal analysis suggest that night-time use of Articulated AFO can promote positive changes in gait parameters of DMD patients, when used before the functional deficit is too advanced., Interpretation: Early daytime and night-time use of Articulated AFO changed gait and minimized typical compensations seen in DMD patients, thus it is recommended in order to prolong gait ability., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To review the hypersensitivity reaction to drugs known as drug reaction with eosinophilia and systemic symptoms (DRESS syndrome), based on a case report. We also intend to discuss the difficulty and importance of disease recognition, since none of the changes is pathognomonic of this disease and failure to identify it may have disastrous consequences for the patient., Method: To describe this case report, in addition to the information collected for clinical assessment, a literature review was performed in the PubMed and Bireme databases in order to retrieve the latest information published in literature on DRESS syndrome., Results: The case of a 20-year old patient is reported. After anamnesis, physical examination and laboratory tests a diagnosis of DRESS syndrome was performed, characterized by rash, hematologic alterations, lymphadenopathy and lesions in target organ. This is a rare syndrome, whose frequency varies according to the drug used and the immune status of the patient, being more often associated with the use of anticonvulsants., Conclusion: The approach and discussion of the topic are of paramount importance, in view of the potential lethality of this treatable syndrome. Recognizing the occurrence of DRESS syndrome and starting treatment as soon as possible is crucial to reduce the risk of mortality and improve prognosis.

The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4(+) and CD8(+) T cells. Higher frequencies of CD4(+) express more than 1 regulatory molecule compared to CD8(+) T cells. Moreover, lower proportions of CD4(+) T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin-3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Despite multidrug therapy, leprosy remains a public health issue. The intradermal Bacillus Calmette-Guérin (BCG) vaccine, Mitsuda test (lepromin skin test), and anti-phenolic glycolipid I (PGL-I) serology are widely used in leprosy studies and have shown great epidemiological value., Methods: This longitudinal study evaluated the relative risks and benefits of these three tools by comparing results observed in household contacts (HHCs) of leprosy patients who developed leprosy with those of HHCs who did not in a population of 2,992 individuals monitored during a 10-year period., Results: Seventy-five (2.5%) new leprosy cases were diagnosed, including 28 (0.9%) co-prevalent cases. Therefore, for the risk-benefit assessment, 47 (1.6%) HHCs were considered as truly diagnosed during follow-up. The comparison between healthy and affected contacts demonstrated that not only did BCG vaccination increase protection, but boosters also increased to 95% relative risk (RR) reduction when results for having two or more scars were compared with having no scars [RR, 0.0459; 95% confidence interval (CI), 0.006-0.338]. Similarly, Mitsuda reactions >7mm in induration presented 7-fold greater protection against disease development compared to reactions of 0-3mm (RR, 0.1446; 95% CI, 0.0566-0.3696). In contrast, anti-PGL-I ELISA seropositivity indicated a 5-fold RR increase for disease outcome (RR, 5.688; 95% CI, 3.2412-9.9824). The combined effect of no BCG scars, Mitsuda reaction of <7mm, and seropositivity to anti-PGL-I increased the risk for leprosy onset 8-fold (RR, 8.109; 95% CI, 5.1167-12.8511)., Conclusions: The adoption of these combined assays may impose measures for leprosy control strategies.

Unlabelled: Little is known about stroke patients' awareness about the warning signs of stroke and its therapeutic time window in Brazil., Method: We interviewed consecutive patients with acute stroke admitted to a terciary public hospital in Brazil. Data collected included demographics, mode of arrival, National Institutes of Health Stroke Scale (NIHSS) scores and knowledge of stroke warning signs and therapeutic time window. Early arrival was defined as within 4.5 hours of symptoms onset., Results: Although 66.2% of patients knew the warning signs of stroke, only 7.8% reported to know that stroke had a limited therapeutic time window. Stroke severity measured by the NIHSS was independently associated with early arrival, but not knowledge of stroke signs and symptoms., Conclusion: Knowledge about stroke symptoms was not a predictor of early arrival.

Using flow cytometry, we evaluated the frequencies of CD4(+) and CD8(+) T cells and Foxp3(+) regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4(+), total Foxp3, and CD4(+) Foxp3(+) cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-β, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-β, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Canine visceral leishmaniasis (CVL) is a severe and fatal systemic chronic inflammatory disease. We investigated the alterations in, and potential associations among, antioxidant enzymes, trace elements and histopathology in CVL. Blood and tissue levels of Cu-Zn superoxide dismutase, catalase and glutathione peroxidase were measured in mixed-breed dogs naturally infected with Leishmania infantum chagasi, symptomatic (n = 19) and asymptomatic (n = 11). Serum levels of copper, iron, zinc, selenium and nitric oxide, and plasma lipid peroxidation were measured. Histological and morphometric analyses were conducted of lesions in liver, spleen and lymph nodes. We found lower blood catalase and glutathione peroxidase activity to be correlated with lower iron and selenium respectively. However, higher activity of Cu-Zn superoxide dismutase was not correlated with the increase in copper and decreased in zinc observed in infected animals compared to controls. Organ tissue was characterized by lower enzyme activity in infected dogs than in controls, but this was not correlated with trace elements. Lipid peroxidation was higher in symptomatic than in asymptomatic and control dogs and was associated with lesions such as chronic inflammatory reaction, congestion, haemosiderin and fibrosis. Systemic iron deposition was observed primarily in the symptomatic dogs showing a higher tissue parasite load. Dogs with symptomatic CVL displayed enhanced LPO and Fe tissue deposition associated with decreased levels of antioxidant enzymes. These results showed new points in the pathology of CVL and might open new treatment perspectives associated with antioxidants and the role of iron in the pathogenesis of CVL., (© 2014 The Authors. International Journal of Experimental Pathology published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).)

Foot drop syndrome is a frequent neurologic condition usually caused by peroneal nerve damage. On rare occasions, foot drop may present as the single neurologic manifestation of intracranial lesions. We presented a 43-year-old man admitted to our hospital with acute weakness in the dorsiflexion of his right foot that appeared 3 days before admission. Brain magnetic resonance imaging diffusion-weighted sequence revealed a small area of restricted diffusion in the left frontal cortex. Three months later, his motor deficit had completely improved (modified Rankin scale score = 0). To our knowledge, this is the second report of sudden isolated foot drop caused by a cortical infarction., (Copyright © 2014 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Objective: Atrial fibrillation is a common arrhythmia that increases the risk of stroke by four- to five-fold. We aimed to establish a profile of patients with atrial fibrillation from a population of patients admitted with acute ischemic stroke or transient ischemic attack using clinical and echocardiographic findings., Methods: We evaluated patients consecutively admitted to a tertiary hospital with acute ischemic stroke or transient ischemic attack. Subjects were divided into an original set (admissions from May 2009 to October 2010) and a validation set (admissions from November 2010 to April 2013). The study was designed as a cohort, with clinical and echocardiographic findings compared between patients with and without atrial fibrillation. A multivariable model was built, and independent predictive factors were used to produce a predictive grading score for atrial fibrillation (Acute Stroke AF Score-ASAS)., Results: A total of 257 patients were evaluated from May 2009 to October 2010 and included in the original set. Atrial fibrillation was diagnosed in 17.5% of these patients. Significant predictors of atrial fibrillation in the multivariate analysis included age, National Institutes of Health Stroke Scores, and the presence of left atrial enlargement. These predictors were used in the final logistic model. For this model, the area under the receiver operating characteristic curve was 0.79. The score derived from the logistic regression analysis was The model developed from the original data set was then applied to the validation data set, showing the preserved discriminatory ability of the model (c statistic = 0.76)., Conclusions: Our risk score suggests that the individual risk for atrial fibrillation in patients with acute ischemic stroke can be assessed using simple data, including age, National Institutes of Health Stroke Scores at admission, and the presence of left atrial enlargement.

Introduction: Lung cancer is the deadliest cancer worldwide. In Portugal, the disease remains the main cause of cancer death in males., Aim: This study aims to evaluate the demographic and clinical characteristics of lung cancer patients diagnosed and treated in northern Portugal hospitals from 2000 to 2010., Patients and Methods: Twelve hospitals in the north of Portugal contributed to this study. The demographic and clinic characteristics of the patients registered in each hospital from 2000 to 2010 and the patterns of their occurrence were analyzed., Results: During an 11-year period (2000-2010), 9767 lung cancer patients were registered in the participating hospitals. Comparing the number of the patients registered in the year 2000 to those registered during 2010, there was a significant increase in lung cancer cases. Females represent only 20% of the total registered lung cancer cases; however, during the study period, the number of female patients increased by 30%. A significant number of the patients, 3117 (48.6%), had poor performance status at presentation. The adenocarcinoma histology became more preponderant over the study period. Most of the patients were diagnosed as stages IIIB or IV: 7206 of 9267 (77.8%). Chemotherapy was the treatment of choice for 3529 (40.4%) patients, whereas surgical treatment was achieved in 1301 (14.9%) cases., Conclusion: A significant number of lung cancer patients have been diagnosed and treated in hospitals in northern Portugal, and the incidence of the disease among females has been increasing. The overwhelming majority of the tumors were diagnosed in advanced stage; nevertheless, surgical treatment was possible in 14.9% of the patients., (Copyright © 2012 Sociedade Portuguesa de Pneumologia. Published by Elsevier España. All rights reserved.)

Purpose: The incidence of pediatric adrenocortical tumors (ACTs) is remarkably high in southern Brazil, where more than 90% of patients carry the germline TP53 mutation R337H. We assessed the impact of early detection of this mutation and of surveillance of carriers., Patients and Methods: Free newborn screening was offered at all hospitals in the state of Paraná. Parents of positive newborns were tested, and relatives in the carrier line were offered screening. Positive newborns and their relatives age < 15 years were offered surveillance (periodic clinical, laboratory, and ultrasound evaluations). ACTs detected by imaging were surgically resected., Results: Of 180,000 newborns offered screening, 171,649 were screened, and 461 (0.27%) were carriers. As of April 2012, ACTs had been diagnosed in 11 of these carriers but in only two neonatally screened noncarriers (P < .001); six patient cases were identified among 228 carrier relatives age < 15 years (total, 19 ACTs). Surveillance participants included 347 (49.6%) of 699 carriers. Tumors were smaller in surveillance participants (P < .001) and more advanced in nonparticipants (four with stage III disease; two deaths). Neonatally screened carriers also had neuroblastoma (n = 1), glioblastoma multiforme (n = 1), choroid plexus carcinoma (n = 2), and Burkitt lymphoma (n = 1). Cancer histories and pedigrees were obtained for 353 families that included 1,704 identified carriers. ACTs were the most frequent cancer among carrier children (n = 48)., Conclusion: These findings establish the prevalence of the TP53 R337H mutation in Paraná state and the penetrance of ACTs among carriers. Importantly, screening and surveillance of heterozygous carriers are effective in detecting ACTs when readily curable.

Background: American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody., Methodology: Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples., Results: The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system)., Conclusions: The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

Background: Infection with parasite protozoa is a long-term health issue in tropical and subtropical regions throughout the world. The Toll-like receptor (TLR) signaling pathway is one of the first-responding defense systems against Leishmania. The aim of this study was to investigate the expression of TLR2 and TLR9 in jejunum and colon and its correlation with CD11c, CD11b, and CD14 receptors used as markers for dendritic cells and macrophages., Methods: Twenty four dogs infected with Leishmania infantum were used in this study. Cytometry was carried out in lamina propria cells from jejunum and colon using markers for TLR2, TLR9, CD11b, CD11c and CD14., Results: Cellular inflammatory exudate was diffuse in the mucosa and submucosa, predominately comprising mononuclear cells: plasma cells, macrophages, and lymphocytes. Despite the parasite load, microscopy showed no erosion was evident in the epithelial mucosa layers. The colon harbored more parasites than the jejunum. Flow cytometry revealed higher frequency of TLR2+ and CD11c+ dendritic cells in the colon than in the jejunum. Conversely, TLR9-expressing cells were more frequent in jejunum. Moreover, frequency of macrophages (CD11b+ and CD14+) expressing simultaneity TLR9 were lower in the colon than in jejunum, while CD11c+ cells predominated in the colon. Despite of the negative ELISA serum results, IL-10 and TNF-α were higher in jejunum than colon of infected animals. However, IL-4 was higher in colon than jejunum of infected animals. A higher expression these cytokines were demonstrated in infected dogs compared to uninfected dogs., Conclusions: There was no correlation between clinical signs and pathological changes and immunological and parasitological findings in the gastrointestinal tract in canine visceral leishmaniasis. However, jejunum showed a lower parasite load with increased frequency and expression of CD11b, TLR9, CD14/CD11b/TLR9 receptors and IL-10 and TNF-α cytokines. Conversely, the colon showed a higher parasite load along with increased frequency and expression of TLR2, CD11c receptors, and IL-4 cytokine. Thus, Leishmania infantum is able to interfere in jejunum increased expression of TLR2, TLR9, CD11b, CD14, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α; and in colon increased expression of CD11c, TLR2, TLR9, CD11b, CD14 e, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α.