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The Zika virus (ZIKV) epidemic declared in Brazil between 2015 and 2016 was associated with an increased prevalence of severe congenital malformations, including microcephaly. The distribution of microcephaly cases was not uniform across the country, with a disproportionately higher incidence in the Northeast region (NE). Our previous work demonstrated that saxitoxin (STX), a toxin present in the drinking water reservoirs of the NE, exacerbated the damaging effects of ZIKV on the developing brain. We hypothesized that the impact of STX might vary among different neural cell types. While ZIKV infection caused severe damages on astrocytes and neural stem cells (NSCs), the addition of STX did not exacerbate these effects. We observed that neurons subjected to STX exposure were more prone to apoptosis and displayed higher ZIKV infection rate. These findings suggest that STX exacerbates the harmful effects of ZIKV on neurons, thereby providing a plausible explanation for the heightened severity of ZIKV-induced congenital malformations observed in Brazil's NE. This study highlights the importance of understanding the interactive effects of environmental toxins and infectious pathogens on neural development, with potential implications for public health policies., (© 2024. The Author(s).)

Mansoa hirsuta is a medicinal plant native to the Brazilian semi-arid region. This approach aimed to investigate the in vitro and in vivo toxicity and anti-inflammatory and analgesic actions of the M. hirsuta fraction (MHF). In vitro cell viability was assessed in 3T3 cells. In vivo, the acute toxicity test, a single dose of the MHF was administered. For the subchronic toxicity test, three doses of were administered for 30 days. Locomotion and motor coordination were assessed using open field and rota-rod. The anti-inflammatory activity was evaluated in carrageenan-induced paw edema and zymosan-induced air-pouch models. Myeloperoxidase (MPO) and total proteins were also measured. The antinociceptive activity MHF was determined using acid acetic-induced abdominal writhing and formalin models. In the cytotoxicity assay, MHF showed no significative impairment of cell viability and in the acute toxicity study, did not cause mortality or signs of toxicity. Repeated exposure to MHF did not cause relevant toxicological changes. The evaluation in the open field test showed that the MHF did not alter the locomotor activity and there was no change in motor coordination and balance of animals. MHF significantly reduced edema, MPO production, the migration of leukocytes and protein leakage. In addition, MHF reduced abdominal writhing and significantly inhibited the first and second stage of the formalin test. The results of this study indicated that MHF has an anti-inflammatory and analgesic potential without causing acute or subchronic toxic effects and it can be a promising natural source to be explored., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Tetragonisca angustula honey was fractioned in a SiO 2 column to furnish three fractions (A-C) in which four hydroxycinnamic acid-Spermidine amides (HCAAs), known as N', N″, N‴-tris-p-coumaroyl spermidine, N', N″-dicaffeoyl, N‴-coumaroyl spermidine, N', N″, N‴-tris-caffeoyl spermidine and N', N″-dicaffeoyl and N‴-feruloyl spermidine were identified in the fractions B and C by electrospray ionization tandem mass spectrometry. A primary culture model previously infected with Neospora caninum (72 h) was used to evaluate the honey fractions (A-C) for two-time intervals: 24 and 72 h. Parasitic reduction ranged from 38% on fraction C (12.5 µg/ml), after 24 h, to 54% and 41% with fractions B and C (25 µg/ml) after 72 h of treatment, respectively. Additionally, HCAAs did not show any cell toxicity for 24 and 72 h. For infected cultures (72 h), the active fractions B (12.5 µg/ml) and C (25 µg/ml) decreased their NO content. In silico studies suggest that HCAAs may affect the parasite's redox pathway and improve the oxidative effect of NO released from infected cells. Here, we presented for the first time, that HCAAs from T. angustula honey have the potential to inhibit the growth of N. caninum protozoa., (© 2021 John Wiley & Sons Ltd.)

Staphylococcus aureus is a Gram-positive bacterium that is considered an important human pathogen. Due to its virulence and ability to acquire mechanisms of resistance to antibiotics, the clinical severity of S. aureus infection is driven by inflammatory responses to the bacteria. Thus, the present study aimed to investigate the modulating role of citral in inflammation caused by S. aureus infection. For this, we used an isolate obtained from a nasal swab sample of a healthy child attending a day-care centre in Vitória da Conquista, Bahia, Brazil. The role of citral in modulating immunological factors against S. aureus infection was evaluated by isolating and cultivating human peripheral blood mononuclear cells. The monocytes were treated with 4%, 2%, and 1% citral before and after inoculation with S. aureus. The cells were analysed by immunophenotyping of monocyte cell surface molecules (CD54, CD282, CD80, HLA-DR, and CD86) and cytokine dosage (IL-1β, IL-6, IL-10, IL-12p70, IL-23, IFN-γ, TGF-β, and TNF-α), and evaluated for the expression of 84 genes related to innate and adaptive immune system responses. GraphPad Prism software and variables with P values < 0.05, were used for statistical analysis. Our data demonstrated citral's action on the expression of surface markers involved in recognition, presentation, and migration, such as CD14, CD54, and CD80, in global negative regulation of inflammation with inhibitory effects on NF-κB, JNK/p38, and IFN pathways. Consequently, IL-1β, IL-6, IL-12p70, IL-23, IFN-γ, and TNF-α cytokine expression was reduced in groups treated with citral and groups treated with citral at 4%, 2%, and 1% and infected, and levels of anti-inflammatory cytokines such as IL-10 were increased. Furthermore, citral could be used as a supporting anti-inflammatory agent against infections caused by S. aureus. There are no data correlating citral, S. aureus, and the markers analysed here; thus, our study addresses this gap in the literature., (© 2021. The Author(s).)

Zika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.

Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease with strong genetic influence, especially upon immune response components. Several cytokines from the toll-like receptors activation pathway display recognized role for RA establishment. However, few studies have verified the role of key mediators such as MYD88 gene and its genetic variants. In the present study, we aim to evaluate the rs6853 functional single-nucleotide variation (SNV) role in RA etiopathogenesis, clinical severity status, and its impact in MYD88 mRNA levels and IL-lβ protein levels. For the association study, a total of 423 RA patients and 346 health individuals, enrolled as control, from Northeast and Southeast Brazil were genotyped using specific Taqman probe. For the gene expression assays, we performed a MYD88 rs6853 genotype-guided monocyte cell culture divided into non-stimulated and lypopolysaccharides (LPS)-stimulated cells from healthy individuals. MYD88 gene expression was measured using primer specifics while IL-1β levels were evaluated by ELISA. We observed that A allele and AA genotype were associated to an increased risk to RA development (OR = 1.60; 95% CI 1.24-2.08; p = 0.0004/OR = 2.83; 95% CI 1.25-6.41; p = 0.0152). The AA genotype exhibited lower MYD88 mRNA levels than GG genotype in non-stimulated monocyte cell culture (FC - 3.83; p = 0.003). Additionally, we verified an increase of IL-1β levels when AA genotype non-stimulated monocytes were compared to AA genotype LPS-stimulates (p = 0.021). In summary, MYD88 rs6853 polymorphism associated to RA development in our Brazilian cohort and showed influence upon MYD88 mRNA levels' expression and IL-lβ production.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility., (©2021 American Association for Cancer Research.)

Propolis is one of the most widely used products in traditional medicine. One of the most prominent types of Brazilian propolis is the red one, whose primary botanical source is Dalbergia ecastaphyllum (L.) Taub. Despite the potential of Brazilian red propolis for developing new products with pharmacological activity, few studies guarantee safety in its use. The objective of this study was the evaluation of the possible toxic effects of Brazilian red propolis and D . ecastaphyllum , as well as the cytotoxicity assessment of the main compounds of red propolis on tumoral cell lines. Hydroalcoholic extracts of the Brazilian red propolis (BRPE) and D . ecastaphyllum stems (DSE) and leaves (DLE) were prepared and chromatographed for isolation of the major compounds. RP-HPLC-DAD was used to quantify the major compounds in the obtained extracts. The XTT assay was used to evaluate the cytotoxic activity of the extracts in the human fibroblast cell line (GM07492A). The results revealed IC 50 values of 102.7, 143.4, and 253.1 μg/mL for BRPE, DSE, and DLE, respectively. The extracts were also evaluated for their genotoxic potential in the micronucleus assay in Chinese hamster lung fibroblasts cells (V79), showing the absence of genotoxicity. The BRPE was investigated for its potential in vivo toxicity in the zebrafish model. Concentrations of 0.8-6.3 mg/L were safe for the animals, with a LC 50 of 9.37 mg/L. Of the 11 compounds isolated from BRPE, medicarpin showed a selective cytotoxic effect against the HeLa cell line. These are the initial steps to determine the toxicological potential of Brazilian red propolis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Genetic and epigenetic factors are considered to be critical for host-parasite interactions. There are limited data on the role of such factors during human infections with Ascaris lumbricoides . Here, we describe the potential role of genetic factors as determinants of the Th2 immune response to A. lumbricoides in Brazilian children. Stool samples were collected from the children to detect A. lumbricoides by microscopy and peripheral blood leukocytes (PBLs) were cultured in whole blood cultures for detection of cytokines (IL-5, IL-10, and IL-13) in vitro . Levels of anti- A. lumbricoides IgE and IgG4 were measured in plasma. DNA was extracted from PBLs and genotyped using Illumina 2.5 Human Omni Beadchip. Candidate genes associated with A. lumbricoides responses were identified and SNVs in these selected genes associated with the Th2 immune response to A. lumbricoides . Haplotype, gene expression, and epigenetic analyses were done to identify potential associations with Th2 immune responses. GWAS on samples from 1,189 children identified WSB1 as a candidate gene, and IL-21R was selected as a biologically relevant linked gene for further analysis. Variants in WSB1 and IL21R were associated with markers of Th2 immune responses: increased A. lumbricoides -specific IgE and IL-5/IL-13 by PBLs from infected compared to uninfected individuals. In infected children, WSB1 but not IL21R gene expression was suppressed and increased methylation was observed in the WSB1 promoter region. This is the first study to show an association between genetic variants in WSB1 and IL21R and Th2 immune responses during A. lumbricoides infections in children. WSB1 / IL21R pathways could provide a potential target for the treatment of Th2-mediated diseases., 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 © 2021 Carneiro, da Silva, Queiroz, Veiga, Oliveira, Carneiro, Pires, da Silva, Sena, Belitardo, Nascimento, Silva, Marques, Costa, Alcantra-Neves, Barreto, Cooper and Figueiredo.)

Extrapulmonary TB (EPTB) occurs with increased frequency in persons with underlying immunodeficiency. Even after recovery from acute illness, differences in immune phenotype and activation persist. Studies defining characteristics of immune responses after recovery from extrapulmonary TB may provide insights into factors that increase TB risk. We performed two case-control studies (in the United States and Brazil) among HIV-seronegative adults with previous EPTB (n = 9; 25), previous pulmonary TB (n = 7; 25), latent M. tuberculosis (Mtb) infection (n = 11; 25), and uninfected TB contacts (n = 10; 25). We assessed the frequency of dual CD4+ interferon-γ and tumor necrosis factor-α responses after stimulation with overlapping Mtb peptides from ESAT-6 or CFP-10, or gamma-irradiated Mtb H37Rv, proliferative responses to Mtb antigens, T-regulatory cell (Treg) frequency and phenotype. In both study populations, individuals with prior EPTB had the highest frequency of intracellular cytokine-producing cells in response to Mtb antigens (p < 0.05; p <.0001). Persons with prior EPTB in Brazil had the highest levels of CD4 proliferation to Mtb antigens (p < 0.0001), and the highest expression of CD39 on Tregs (p < 0.0001). Individuals with treated EPTB maintained high frequencies of Mtb-specific memory responses and active Treg cells, suggesting that susceptibility to EPTB occurs despite the ability to develop and maintain enhanced adaptive immune responses., 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. The handling editor declared a past co-authorship with the authors BA and TS., (Copyright © 2020 Barreto-Duarte, Sterling, Fiske, Almeida, Nochowicz, Smith, Barnett, Warren, Blackman, Lapa e Silva, Andrade and Kalams.)

The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Poorly controlled diabetes mellitus leads to several comorbidities, including susceptibility to infections. Hyperglycemia increases phagocyte responsiveness, however immune cells from people with diabetes show inadequate antimicrobial functions. We and others have shown that aberrant production of leukotriene B 4 (LTB 4 ) is detrimental to host defense in models of bacterial infection. Here, we will unveil the consequences of high glucose in the outcome of Leishmania braziliensis skin infection in people with diabetes and determine the role of LTB 4 in human phagocytes. We show that diabetes leads to higher systemic levels of LTB 4 , IL-6 and TNF-α in cutaneous leishmaniasis. Only LTB 4 correlated with blood glucose levels and healing time in diabetes comorbidity. Skin lesions of people with leishmaniasis and diabetes exhibit increased neutrophil and amastigote numbers. Monocyte-derived macrophages from these individuals showed higher L. braziliensis loads, reduced production of Reactive Oxygen Species and unbalanced LTB 4 /PGE 2 ratio. Our data reveal a systemic inflammation driven by diabetes comorbidity in opposition to a local reduced capacity to resolve L. braziliensis infection and a worse disease outcome.

Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans , we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1 Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis. IMPORTANCE Cryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages., (Copyright © 2020 Frazão et al.)

Caatinga flora which are found in a poor Brazilian region contain a substantial number of endemic taxa with biomedical and social importance for regional communities. This study examined the antioxidant and cytotoxic potential of 35 samples (extracts/fractions) from 12 Caatinga species and determined the antiproliferative and genotoxic action of dichloromethane fraction from Mimosa caesalpiniifolia stem bark (DC-Mca) on human and vegetal cells. Samples were assessed for chemopreventive ability, toxic effects on Artemia salina shrimp as well as cytotoxicity on tumor cell lines and erythrocytes. DC-Mca was also tested with respect to antiproliferative and genotoxic effects upon normal leukocytes and meristematic cells from A. cepa roots. Some extracts reduced free radical levels >95% and 7 samples exhibited a lethal concentration (LC) 50  < 100 µg/ml upon Artemia salina larvae. Eight samples displayed in vitro antitumor effects and three produced hemolysis. Data also demonstrated the pharmacological significance of bioactive extracts from Brazilian semi-arid region. There was no significant relationship between antioxidant, toxic, and antiproliferative activities, and that these properties were dependent upon the extractant. DC-Mca contained betulinic acid as main compound (approximately 70%), which showed higher (1) cytotoxic activity on cancer cell lines and dividing leukocytes, (2) reduced mitotic index of Allium cepa roots, and (3) induced cell cycle arrest and chromosomal bridges, thereby providing native promising sources for phytotherapy development., Abbreviations: ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); AcOH: ethyl acetate; ANOVA: analysis of variance; SUS: Brazilian Unified Health System; DC-Mca: dichloromethane fraction from Mimosa caesalpiniifolia stem bark; DMSO: dimethylsulfoxide; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EC 50 : effective concentration 50%; EtOAc: ethyl acetate; FDA: Food and Drug Administration; GC-Qms: gas chromatograph quadrupole mass spectrometer; GI: genotoxic index; HCT-116: colon carcinoma line; HL-60: promyelocytic leukemia line; HPLC: high-performance liquid chromatography; HRAPCIMS: high resolution atmospheric pressure chemical ionization mass spectrum; IC 50 : inhibitory concentration 50%; LC 50 : lethal concentration 50%; MeOH = methyl alcohol; MI: mitotic index; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; MutI: mutagenic index; OVCAR-8 = ovarian carcinoma line; PBMC: peripheral blood mononuclear cells; RPMI-1640: Roswell Park Memorial Institute medium; SF-295: glioblastoma line; TEAC: trolox equivalent antioxidant capacity; TLC: thin-layer chromatography; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

Background: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states., Results: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors., Conclusion: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.

Serum soluble Fas (sFas) levels are associated with erythropoietin (Epo) hyporesponsiveness in patients with chronic kidney disease (CKD). Whether sFas could predict the need for erythropoiesis-stimulating agent (ESA) usage and its influence in erythropoiesis remain unclear. We evaluated the relation between sFas and ESA therapy in patients with CKD with anemia and its effect on erythropoiesis in vitro. First, we performed a retrospective cohort study with 77 anemic patients with nondialysis CKD. We performed in vitro experiments to investigate whether sFas could interfere with the behavior of hematopoietic stem cells (HSCs). HSCs were isolated from umbilical cord blood and incubated with recombinant sFas protein in a dose-dependent manner. Serum sFas positively correlated with Epo levels ( r  = 0.30, P = 0.001) but negatively with hemoglobin ( r  = -0.55, P < 0.001) and glomerular filtration rate ( r  = -0.58, P < 0.001) in patients with CKD at baseline. Elevated sFas serum levels (4,316 ± 897 vs. 2,776 ± 749, P < 0.001) with lower estimated glomerular filtration rate (26.2 ± 10.1 vs. 33.5 ± 14.3, P = 0.01) and reduced hemoglobin concentration (11.1 ± 0.9 vs. 12.5 ± 1.2, P < 0.001) were identified in patients who required ESA therapy compared with patients with non-ESA. Afterward, we detected that the sFas level was slight correlated with a necessity of ESA therapy in patients with nondialysis CKD and anemia. In vitro assays demonstrated that the erythroid progenitor cell frequency negatively correlated with sFas concentration ( r  = -0.72, P < 0.001). There was decreased erythroid colony formation in vitro when CD34 + HSCs were incubated with a higher concentration of sFas protein (1.56 ± 0.29, 4.33 ± 0.53, P < 0.001). Our findings suggest that sFas is a potential predictor for ESA therapy in patients with nondialysis CKD and that elevated sFas could affect erythropoiesis in vitro.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to evaluate the effect of mineral trioxide aggregate (MTA) and Brazilian propolis on the cell viability, mineralization, anti-inflammatory ability, and migration of human dental pulp cells (hDPCs). The cell viability was evaluated with CCK-8 kit after 1, 5, 7, and 9 days. The deposition of calcified matrix and the expression of osteogenesis-related genes were evaluated by Alizarin Red staining and real-time PCR after incubation in osteogenic medium for 21 days. The expression of inflammation-related genes in cells was determined after exposure to 1 μg/mL LPS for 3 h. Finally, the numbers of cells that migrated through the permeable membranes were compared during 15 h. Propolis and MTA significantly increased the viability of hDPCscompared to the control group on days 7 and 9. In the propolis group, significant enhancement of osteogenic potential and suppressed expression of IL-1β and IL-6 was observed after LPS exposure compared to the MTA and control groups. The number of migration cells in the propolis group was similar to that of the control group, while MTA significantly promoted cell migration. Propolis showed comparable cell viability to that of MTA and exhibited significantly higher anti-inflammatory and mineralization promotion effects on hDPCs.

Two one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of Zika virus (ZIKV) were developed, based on two different primer design approaches: (1) open source, based on a combination of sequence diversity clustering (phylogeny and principal component analysis) and LAVA algorithm, using 45 whole genome ZIKV sequences retrieved from the National Center for Biotechnology Information (NCBI) database; (2) standard software for LAMP primer design (Primer Explorer V4), using 59 sequences of the ZIKV 3' UTR. The assays were firstly evaluated with External Quality Assessment panels from INSTAND e.V. (Germany) and EVD-LabNet (The Netherlands) including 4 and 12 unknown samples, respectively, and secondly, with 9 human, mosquito, and monkey ZIKV isolates from Africa (Senegal, Ivory Coast, and Uganda) and America (Brazil). The limit of detection as determined by probit analysis was 181 molecules for both RT-LAMP assays, and 100% reproducibility in the assays was obtained for 10 3  molecules (4/8 repetitions were positive for 10 2  molecules). Both assays were specific, amplifying only ZIKV RNA and not cross-detecting other arboviruses included in this study.

Molecular detection of Zika virus (ZIKV) is a key element of outbreak management. Multiple PCR and isothermal ZIKV assays targeting different ZIKV sequences have been published. In this study, we compared a qRT-PCR, 2 RT-LAMP assays (based on different primer design approaches), and an RT-RPA for the detection of African and Asian/American lineages of ZIKV isolates from human, mosquito, and monkey. Results showed that RT-LAMP detected 100% of samples with a time threshold (Tt) of 18.01 ± 11.71 min while qRT-PCR detected 88.88% of samples with a Tt of 58.30 ± 16.58 min and RT-RPA 50% of samples with a Tt of 3.70 ± 0.44 min.

Background: Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV., Methods: PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry., Results: We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3 + CD4 + T cells, CD3 - CD19 + B cells and CD3 + CD8 + T cells being, respectively, the most frequently infected subpopulations, followed by CD14 + monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells., Conclusions: These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Pereskia aculeata Miller, known worldwide as ora-pro-nobis, is a highly nutritive species of the Cactaceae family from the Brazilian Atlantic Forest. In this work, we report inedited information on the phenolic profile of P. aculeata leaves, besides a broad study of their antioxidant potential using a set of five different methods. A total of ten phenolic compounds were identified, such as two phenolic acids (caffeic acid derivatives) and eight flavonoids (quercetin, kaempferol and isorhamnetin glycoside derivatives). Caftaric acid was the extract's major phenolic constituent, accounting for more than 49% of the phenolic content, followed by quercetin-3-O-rutinoside (14.99%) and isorhamnetin-O-pentoside-O-rutinoside (9.56%). Overall, the ora-pro-nobis leaf extract showed relevant values of antioxidant capacity, with higher activities than the Trolox in the DPPH and ABTS trials. The antimicrobial activity exhibited by the extract against both Gram-positive and Gram-negative bacteria suggests the presence of a broad spectrum of phytochemicals with antibiotic activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The MeOH extract from leaves of Saururus cernuus L. (Saururaceae) displayed in vitro activity against trypomastigote forms of T. cruzi (100% of parasite death at 200 μg/mL), suggesting the presence of bioactive compounds. Thus, the bioactivity-guided fractionation was carried out, leading to the isolation of three related neolignan derivatives, identified as threo-austrobailignan-5 (1), threo-austrobailignan-6 (2), and threo-dihydroguaiaretic acid (3). Anti-T. cruzi activity of compounds 1-3 was performed against cell-derived trypomastigotes and intracellular amastigotes. Additionally, the mammalian cytotoxicity was investigated using NCTC cells. Compound 2 was the most effective against extracellular trypomastigotes with IC 50 of 3.7 μM, while compound 3 showed activity in both clinically relevant forms of the parasite, trypomastigotes and amastigotes, with IC 50 values of 7.0 and 16.2 μM, respectively. However, the structurally related compound 1 was inactive. Based on these results, compounds 2 and 3 were selected to evaluate the mechanism of cellular death. Compound 2 induced alteration in the plasma membrane permeability and consequently in the ROS levels after 120 min of incubation. By using flow cytometry and fluorescence microscopy, compound 3 showed alterations in the mitochondrial membrane potential (ΔΨ m ) of trypomastigotes. Considering the promising chemical and biological properties of neolignans 2 and 3, these compounds could be used as starting points to develop new lead compounds for Chagas disease., (Copyright © 2019. Published by Elsevier B.V.)

Zika virus (ZIKV) infection during pregnancy is associated with microcephaly, a congenital malformation resulting from neuroinflammation and direct effects of virus replication on the developing central nervous system (CNS). However, the exact changes in the affected CNS remain unknown. Here, we show by transcriptome analysis (at 48 h post-infection) and multiplex immune profiling that human induced-neuroprogenitor stem cells (hiNPCs) respond to ZIKV infection with a strong induction of type-I interferons (IFNs) and several type-I IFNs stimulated genes (ISGs), notably cytokines and the pro-apoptotic chemokines CXCL9 and CXCL10. By comparing the inflammatory profile induced by a ZIKV Brazilian strain with an ancestral strain isolated from Cambodia in 2010, we observed that the response magnitude differs among them. Compared to ZIKV/Cambodia, the experimental infection of hiNPCs with ZIKV/Brazil resulted in a diminished induction of ISGs and lower induction of several cytokines (IFN-α, IL-1α/β, IL-6, IL-8, and IL-15), consequently favoring virus replication. From ZIKV-confirmed infant microcephaly cases, we detected a similar profile characterized by the presence of IFN-α, CXCL10, and CXCL9 in cerebrospinal fluid (CSF) samples collected after birth, evidencing a sustained CNS inflammation. Altogether, our data suggest that the CNS may be directly affected due to an unbalanced and chronic local inflammatory response, elicited by ZIKV infection, which contributes to damage to the fetal brain.

Background: Residual HIV-1 replication among individuals under antiretroviral therapy (ART) relates to HIV micro-inflammation., Objectives: To determine the levels of residual HIV replication markers among distinct subgroups of antiretroviral-treated individuals., Methods: One hundred sixteen patients were distributed into 5 treatment groups: first-line suppressive ART with a non-nucleoside analog reverse-transcriptase inhibitor (NNRTI) (n = 26), first-line suppressive ART with boosted protease inhibitors (PI-r) (n = 25), salvage therapy using PI-r (n = 27), salvage therapy with PI-r and raltegravir (n = 22) and virologic failure (n = 16). Episomal and total DNA quantitation was evaluated. ELISA was used for HIV antibody and LPS quantitation., Results: Episomal DNA was positive in 26% to 38% of individuals under suppressive ART, and it was higher among individuals experiencing ART virologic failure (p = 0.04). The HIV proviral load was higher among patients with detectable episomal DNA (p = 0.01). Individuals receiving initial PI-r treatment presented lower HIV antibody (p = 0.027) and LPS (p = 0.029) levels than individuals receiving NNRTI. There was a negative correlation between episomal DNA quantitation and the duration of suppressive ART (p = 0.04), CD4+ T-cell count (p = 0.08), and CD8+ T-cell count (p = 0.07)., Conclusions: Residual HIV replication has been inferred among individuals under suppressive ART according to episomal DNA detection. Residual replication may decrease with longer periods of suppressive ART and higher levels of CD4+ and CD8+ T cells. The relationship between episomal DNA and total DNA suggests there is a replenishment of the proviral reservoir with impacts on HIV persistence. Lower antibody and LPS levels among patients with initial PI-r ART suggest these regimens may more effectively suppress HIV and have a higher capacity to decrease the HIV antigenic component., Competing Interests: The authors have declared that no competing interests exist.

Eugenia dysenterica is a Brazilian tree investigated for its properties and bioactive compounds, which are believed to have both pharmacological and phytochemical therapeutic effects. The leaves of this tree contain tannins, flavonoids, terpenes, and saponins, with reportedly beneficial effects to the human body. Despite these therapeutic applications, its effects have never been tested on oral tissues. Therefore, the aim of the present study was to evaluate the cytotoxic and antioxidant effects and the anti-inflammatory and repair properties of the acetone fraction of E. dysenterica on primary culture of human gingival fibroblasts and on the immortalized murine macrophage cell line (RAW 264.7). For this purpose, a metabolic activity assay, a wound healing assay, a nitric oxide assay, and RT-qPCR were performed. The assays revealed a cytoprotective effect of this plant, suggested by the increase in the expression of SOD1 and NRF2. An antioxidant potential effect was observed in the DPPH• assay. However, the fraction of E. dysenterica did not show anti-inflammatory activity. In conclusion, Eugenia dysenterica may promote cytoprotection when associated with chlorhexidine digluconate because of its antioxidant effect. However, additional studies are necessary on other human dental tissues using other parts of the plant in order to develop a possible mouthwash to assist patients with oral disorders.

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.

B-cells mediate humoral adaptive immune response via the production of antibodies and cytokines, and by inducing T-cell activation. These functions can be attributed to distinct B-cell subpopulations. Infection with Trypanosoma cruzi , the causative agent of Chagas disease, induces a polyclonal B-cell activation and lytic antibody production, critical for controlling parasitemia. Individuals within the chronic phase of Chagas disease may remain in an asymptomatic form (indeterminate), or develop severe cardiomyopathy (cardiac form) that can lead to death. Currently, there is no effective vaccine to prevent Chagas disease, and no treatment to halt the development of the cardiomyopathy once it is installed. The pathology associated with cardiac Chagas disease is a result of an inflammatory reaction. Thus, discovering characteristics of the host's immune response that favor the maintenance of favorable heart function may unveil important immunotherapeutic targets. Given the importance of B cells in antibody production and parasite control, we investigated T. cruzi- derived antigenic fractions responsible for B-cell activation and whether frequencies and functional characteristics of B-cell subpopulations are associated with different clinical outcomes of human Chagas disease. We stimulated cells from indeterminate (I) and cardiac (C) Chagas patients, as well as non-infected individuals (NI), with T. cruzi -derived protein- (PRO), glycolipid- (GCL) and lipid (LIP)-enriched fractions and determined functional characteristics of B-cell subpopulations. Our results showed that the frequency of B-cells was similar amongst groups. PRO, but not GCL nor LIP, led to an increased frequency of B1 B-cells in I, but not C nor NI. Although stimulation with PRO induced higher TNF expression by B1 B-cells from C and I, as compared to NI, it induced expression of IL-10 in cells from I, but not C. Stimulation with PRO induced an increased frequency of the CD11b + B1 B-cell subpopulation, which was associated with better cardiac function. Chagas patients displayed increased IgM production, and activation of gamma-delta T-cells, which have been associated with B1 B-cell function. Our data showed that PRO activates CD11b + B1 B-cells, and that this activation is associated with a beneficial clinical status. These findings may have implications in designing new strategies focusing on B-cell activation to prevent Chagas disease cardiomyopathy.

Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low action/secretion of the hypothalamic gonadotrophin-releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype-genotype CHH analysis using an in-house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin-releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS-7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild-type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype-genotype correlation in CHH., (© 2018 British Society for Neuroendocrinology.)

Background: Several species of Aspidosperma plants are referred to as remedies for the treatment of malaria, especially Aspidosperma nitidum. Aspidosperma pyrifolium, also a medicinal plant, is used as a natural anti-inflammatory. Its fractionated extracts were assayed in vitro for activity against malaria parasites and for cytotoxicity., Methods: Aspidosperma pyrifolium activity was evaluated against Plasmodium falciparum using extracts in vitro. Toxicity towards human hepatoma cells, monkey kidney cells or human monocytes freshly isolated from peripheral blood was also assessed. Anti-malarial activity of selected extracts and fractions that presented in vitro activity were tested in mice with a Plasmodium berghei blood-induced infection., Results: The crude stem bark extract and the alkaloid-rich and ethyl acetate fractions from stem extract showed in vitro activity. None of the crude extracts or fractions was cytotoxic to normal monkey kidney and to a human hepatoma cell lines, or human peripheral blood mononuclear cells; the MDL 50 values of all the crude bark extracts and fractions were similar or better when tested on normal cells, with the exception of organic and alkaloidic-rich fractions from stem extract. Two extracts and two fractions tested in vivo caused a significant reduction of P. berghei parasitaemia in experimentally infected mice., Conclusion: Considering the high therapeutic index of the alkaloidic-rich fraction from stem extract of A. pyrifolium, it makes the species a candidate for further investigation aiming to produce a new anti-malarial, especially considering that the active extract has no toxicity, i.e., no mutagenic effects in the genototoxicity assays, and that it has an in vivo anti-malarial effect. In its UPLC-HRMS analysis this fraction was shown to have two major components compatible with the bisindole alkaloid Leucoridine B, and a novel compound, which is likely to be responsible for the activity against malaria parasites demonstrated in in vitro tests.

Hypertension, a chronic non-transmissible multifactorial condition, it is highly frequent in Brazil, affecting about 32.5% of the population over 25 years of age. It is characterized by the sustained increase in systolic and diastolic blood pressure levels above 140 mmHg and 90 mmHg, respectively. It is the major aggravating factor in cardiovascular complications and the appearance of other comorbidities. Aiming to promote greater adherence to treatment and improve the population's access to basic medicament, in 2004 the Federal Government created the Programa Farmácia Popular do Brasil (PFPB); partnership with private institutions that provides the population with medicament to control hypertension, free of charge or subsidized at up to 90% of the value. The PFPB distributes the anti-hypertensives atenolol, captopril, enalapril, hydrochlorothiazide, losartan and propranolol. In this way, this work aims to evaluate the genotoxic potential of antihypertensives in human lymphocytes and macrophages, since they are widely used drugs and with few studies about their genotoxicological safety. The tests were developed from cell cultures treated with five different antihypertensive concentrations, all based on plasma peaks, evaluating cell viability, DNA damage index and DNA double strand breakdown. The results show that, as the concentration of captopril and enalapril maleate increased, cell viability decreased. In addition, a DNA damage was observed with the use Captopril and Enalapril in the higher concentrations. Hydrochlorothiazide also caused DNA damage in the five doses tested. Regarding the breaking of double strands of DNA, all the compounds showed increased ruptures. This decrease in dsDNA is dose dependent for all compounds tested. The set of results shows that the use although frequent still requires care and greater knowledge. In general, the antihypertensive drugs that proved to be safer in relation to the genetic damage tested were Losartan and Propranolol., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: This in vivo research investigated whether pulp treatments using formocresol for 7 days would cause mutagenic changes in children's lymphocytes., Materials and Methods: The mutagenicity was tested in lymphocyte cultures established from the peripheral blood of children living in Brazil. The samples consisted of 2000 cells from teeth undergoing formocresol pulpotomies in which the formocresol pellet was sealed in the primary tooth for 7 days. It was removed on the seventh day, the base was placed, and the tooth was restored. Two venous blood samples (6-8 ml) were collected from each child; the first was prior to pulp therapy, and the second was 7 days later. Two thousand metaphases were analyzed. The level of significance adopted for the statistics was P < 0.05, and a random effects meta-analysis was performed combining this and two previous studies., Results: There was no significant difference found in the metaphase analysis between the blood samples taken before and after the pulpotomy treatment (Wilcoxon signed rank test); however, the meta-analysis showed a significant difference between the combined studies., Conclusions: This study did not reveal any mutagenic effects, but based on the combined meta-analysis, we recommend the careful use of formocresol., Clinical Relevance: This research helps to bring scientific evidence of the safe use of formocresol in deciduous pulpotomy treatments.

In this study, we aimed to evaluate the immunomodulatory effects of crude leaf extracts from Piper gaudichaudianum Kunth, P. arboreum Aub., P. umbellata L., P. fuligineum Kunth, and Peperomia obtusifolia A. Dietr. on an in vitro model of inflammatory response. The crude extracts were previously obtained by maceration of the leaves. The half-maximal inhibitory concentration was determined by the MTT assay using human peripheral blood mononuclear cells. Human monocytes were simultaneously challenged with each crude extract and lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, to induce a strong inflammatory response. After 24 h of incubation, cell-free supernatants were used for evaluating the mediators involved in inflammation: H2O2, TNF-α, IL-8, IL-6, IL-1β, IL-10, IL-12, FGF-b, and TGF-β1. We also compared the results with the effects of ketoprofen, a well-known anti-inflammatory drug. The P. gaudichaudianum crude extract downmodulated the production of H2O2, IL-1β, IL-6, IL-8, and TGF-β1 by LPS-stimulated monocytes; P. arboreum, IL-1β, IL-6, IL-8, and TNF-α; P. umbellata and P. fuligineum, H2O2, IL-1β, IL-6, IL-8, IL-10, and TNF-α; and P. obtusifolia, H2O2, IL-6, IL-8, IL-10, and TNF-α. In general, the crude leaf extracts amplified the anti-inflammatory response when compared with ketoprofen, particularly reducing the production of IL-8, a mediator involved in neutrophil recruitment during tissue damage. Thus, the crude leaf extracts of P. gaudichaudianum, P. arboreum, P. umbellata, P. fuligineum, and Peperomia obtusifolia elicited an anti-inflammatory response against LPS-challenged monocytes. These findings show the anti-inflammatory properties of these crude leaf extracts and offer new perspectives for their use in the treatment of inflammatory diseases., Competing Interests: The authors have declared that no competing interests exist.

Fogo Selvagem (FS), the endemic form of pemphigus foliaceus, is mediated by pathogenic IgG4 autoantibodies against the amino-terminal extracellular cadherin domain of the desmosomal cadherin desmoglein 1 (Dsg1). Here we define the detailed epitopes of these pathogenic antibodies. Proteolytic footprinting showed that IgG4 from 95% of FS donor sera (19/20) recognized a 16-residue peptide (A 129 LNSMGQDLERPLELR 144 ) from the EC1 domain of Dsg1 that overlaps the binding site for an adhesive-partner desmosomal cadherin molecule. Mutation of Dsg1 residues M 133 and Q 135 reduced the binding of FS IgG4 autoantibodies to Dsg1 by ∼50%. Molecular modeling identified two nearby EC1 domain residues (Q 82 and V 83 ) likely to contribute to the epitope. Mutation of these residues completely abolished the binding of FS IgG4 to Dsg1. Bead aggregation assays showed that native binding interactions between Dsg1 and desmocollin 1 (Dsc1), which underlie desmosome structure, were abolished by Fab fragments of FS IgG4. These results further define the molecular mechanism by which FS IgG4 autoantibodies interfere with desmosome structure and lead to cell-cell detachment, the hallmark of this disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8 + and CD4 + T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n  = 110) or cardiopathic (mild, B1, n  = 163; severe, C, n  = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P -value = 0.04) and T carriers (OR = 0.5, P -value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1 + CD8 + T cells and CD14 + macrophages were decreased, while frequencies of CCR5 + cells were increased. Importantly, CCR1 + CD14 + macrophages were mainly IL-10 + , while CCR5 + cells were mostly TNF + . CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5 -/- mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1 + CD8 + T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1 + cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5 µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

Cutaneous leishmaniasis (CL) is the most common clinical form of American tegumentary leishmaniasis caused by Leishmania ( Viannia ) braziliensis . CL is associated with a strong Th1 immune response. This exacerbated inflammatory response is correlated with severity of disease and delays the healing time of the ulcer. The fourth-generation immucillin derivative (DI4G), a potent inhibitor of purine nucleoside phosphorylase, has been proposed as a promising agent in the treatment of diseases associated with T cell activation. Herein, we evaluated the in vitro immunomodulatory activity of DI4G in cells of patients presenting with CL. Peripheral blood mononuclear cells (PBMC) from CL patients were stimulated with soluble leishmania antigen (SLA), in the presence or absence of DI4G, and IFN- γ , TNF, CXCL9, and CXCL10 levels were determined by ELISA. Lymphocyte proliferation in the presence or absence of DI4G was also evaluated, using flow cytometry. DI4G was able to decrease ( p < 0.05) IFN- γ production but did not change the TNF, CXCL9, and CXCL10 levels. DI4G decreased ( p < 0.05) the lymphoproliferative response mediated by CD8 + T cells, but not that by CD4 + T cells. DI4G is able to attenuate the exaggerated immune response in CL, exhibiting immunomodulatory activity in IFN- γ production and in CD8 + T cell proliferation.

Mansoa hirsuta (Bignoniaceae) is a native plant from caatinga in Brazilian semiarid. This plant has been locally used as antimicrobial and hypoglycemiant agents, but their action mechanisms and toxicity remain largely unknown. Therefore, we evaluated the composition and antioxidant, cytoprotective and hypoglycemiant effects of raw extract, fractions and compounds from leaves of M. hirsuta. The cytogenotoxic effects of ursolic and oleanolic acids, the main phytotherapic components of this plant, were assessed. The raw extract and fractions presented steroids, saponins, flavonols, flavanonols, flavanones, xanthones, phenols, tannins, anthocyanins, anthocyanidins and flavonoids. The ethyl acetate fraction inhibited efficiently the cascade of lipid peroxidation while the hydroalcoholic fraction was richer in total phenols and more efficient in capturing 2,2-diphenyl-1-picrylhydrazyl (·DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS·+) radicals. The isolated fraction of M. hirsuta also inhibited the α-amylase activity. Cytotoxic effects were absent in both raw extract and fractions while ursolic+oleanolic acids were efficient in protecting cells after exposure to hydrogen peroxide. Moreover, this mixture of acid shad no significant interference on the mitotic index and frequency of nuclear and/or chromosomal abnormalities in Allium cepa test. Therefore, M. hirsuta represents a potential source of phytochemicals against inflammatory and oxidative pathologies, including diabetes.

Bloodstream infections caused by Candida species are responsible for high morbidity and mortality, and diabetes mellitus (DM) is an important underlying disease in candidemia episodes. Although DM patients show an enhanced proinflammatory profile, they are highly susceptible to mycobacterial and mycotic infections. Attempting to understand this paradox, we investigated if imbalanced macrophage and dendritic cell (DC) activations could be associated to high incidence and/or severity of Candida albicans infection in the hypoinsulinemia-hyperglycemia (HH) milieu. HH alloxan-induced mice were infected with C. albicans and peritoneal aderent phagocytes were co-cultured with or without lipopolyssaccharide or heat-killed C. albicans, and the production of cytotoxic metabolites, cytokines, and chemokines was evaluated. We also evaluated the surface expression of MHC-II and CD86 in splenic DCs. Our findings showed that both uninfected and C. albicans-infected HH mice showed less production of CCL2 and reduced expression of CD86 by peritoneal phagocytes and splenic DCs, respectively.

Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development., (Copyright © 2016, American Association for the Advancement of Science.)

Background: The anti-malarials quinine and artemisinin were isolated from traditionally used plants (Cinchona spp. and Artemisia annua, respectively). The synthetic quinoline anti-malarials (e.g. chloroquine) and semi-synthetic artemisinin derivatives (e.g. artesunate) were developed based on these natural products. Malaria is endemic to the Amazon region where Plasmodium falciparum and Plasmodium vivax drug-resistance is of concern. There is an urgent need for new anti-malarials. Traditionally used Amazonian plants may provide new treatments for drug-resistant P. vivax and P. falciparum. Herein, the in vitro and in vivo antiplasmodial activity and cytotoxicity of medicinal plant extracts were investigated., Methods: Sixty-nine extracts from 11 plant species were prepared and screened for in vitro activity against P. falciparum K1 strain and for cytotoxicity against human fibroblasts and two melanoma cell lines. Median inhibitory concentrations (IC50) were established against chloroquine-resistant P. falciparum W2 clone using monoclonal anti-HRPII (histidine-rich protein II) antibodies in an enzyme-linked immunosorbent assay. Extracts were evaluated for toxicity against murine macrophages (IC50) and selectivity indices (SI) were determined. Three extracts were also evaluated orally in Plasmodium berghei-infected mice., Results: High in vitro antiplasmodial activity (IC50 = 6.4-9.9 µg/mL) was observed for Andropogon leucostachyus aerial part methanol extracts, Croton cajucara red variety leaf chloroform extracts, Miconia nervosa leaf methanol extracts, and Xylopia amazonica leaf chloroform and branch ethanol extracts. Paullinia cupana branch chloroform extracts and Croton cajucara red variety leaf ethanol extracts were toxic to fibroblasts and or melanoma cells. Xylopia amazonica branch ethanol extracts and Zanthoxylum djalma-batistae branch chloroform extracts were toxic to macrophages (IC50 = 6.9 and 24.7 µg/mL, respectively). Andropogon leucostachyus extracts were the most selective (SI >28.2) and the most active in vivo (at doses of 250 mg/kg, 71% suppression of P. berghei parasitaemia versus untreated controls)., Conclusions: Ethnobotanical or ethnopharmacological reports describe the anti-malarial use of these plants or the antiplasmodial activity of congeneric species. No antiplasmodial activity has been demonstrated previously for the extracts of these plants. Seven plants exhibit in vivo and or in vitro anti-malarial potential. Future work should aim to discover the anti-malarial substances present.

Background: Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection., Methodology and Principal Findings: Here we investigated the contribution of galectin-1 (Gal-1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL-1 cardiac cells to Gal-1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal-1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL-1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal-1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal-1 to the cell surface. Consistent with these data, Gal-1 deficient (Lgals1-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain., Conclusion/significance: Our results indicate that Gal-1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.

Introduction: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility., Objective: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17)., Methods: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells., Results: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group., Conclusions: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.

Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)