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The corn leafhopper, Dalbulus maidis (Hemiptera: Cicadellidae), spreads maize stunt pathogens and requires timely and effective crop protection. We determined the interaction between maize phenology and the vector feeding/infection period by stunt pathogens with the residual efficacy of neonicotinoid insecticidal seed treatments. Greenhouse- and field-grown maize plants, seed-treated with clothianidin or imidacloprid insecticides, were infested during seven growth stages with corn leafhoppers reared under controlled conditions on maize plants displaying infection symptoms by both spiroplasma (corn stunt spiroplasma, Spiroplasma kunkelii) and phytoplasma (maize bushy phytoplasma) pathogens.In the greenhouse and field settings, seed treatment reduced the stunt disease symptoms and corn yield loss during the VE-V4 maize growth stages and caused no phytotoxicity. The neonicotinoid seed treatment reduced 20-60% of the yield losses from the corn stunt disease until the V4 growth stage. Infestation by infective corn leafhoppers in the V12 maize growth stage caused a 25-30% yield loss irrespective of seed treatment, yet no stunt disease symptom was evident. Nonetheless, corn yield losses and visual stunt symptoms as rated by a nine-category ordinal scale were strongly correlated (r = 0.79, P 0.01).These results reinforce that maize plants are more susceptible to leafhopper stunt disease during the VE-V4 growth stages (emergence to the fourth-leaf stage). Seed treatment helps reduce the damage in the early growth stages (VE-V2), although supplemental control measures depending on leafhopper population density may be needed from VE-V12 to protect yield losses from the maize stunt condition. © 2021 Society of Chemical Industry.

BACKGROUND The highly polyphagous and invasive fall armyworm (FAW, Spodoptera frugiperda) can feed on different plant parts of host crops, damaging whorls and stalks in early maize growth stages. Systemic insecticide seed treatment (IST) could minimize this damage, although the residual efficacy may vary with the plant tissue damaged. Using damage rating scales and artificial infestation in controlled conditions, we determined the potential of IST against FAW attacking maize whorl leaves or the stalk base. RESULTS Chlorantraniliprole, cyantraniliprole, or thiodicarb+imidacloprid IST similarly killed >80% FAWs for one or two weeks after plant emergence depending on the plant tissue attacked. The residual efficacy (i.e., time after plant emergence sustaining >80% larval mortality) lasted from the first to the eleventh day (VE-V3 maize growth stages), while for cutworm on the maize stalk base, it lasted 3-7 days after plant emergence (V1-V2 stages). In terms of damage, the ISTs lasted 15 days after emergence (V4 stage) for FAW on whorl leaves and 10 days (V3 stage) for FAW feeding on the stalk base. The larvae surviving on the seed-treated plants underwent sublethal effects in growth and development, reducing insect fitness. CONCLUSION Diamide or carbamate+neonicotinoid seed treatments kill FAW larvae on maize whorls or stalks in favorable edaphoclimatic and insecticide-susceptibility conditions. The cumulative impacts of systemic IST on above-ground insect pests go beyond mortality. The ISTs studied can be valuable against FAW in maize, for instance, to help protect varieties that may not express sufficient insect resistance in maize early growth stages. This article is protected by copyright. All rights reserved.

The objectives of this study were to quantify the virulence of four entomopathogenic fungal species to pupae of Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) and to determine the potential to combine entomopathogenic fungi (EPFs) and entomopathogenic nematodes (EPNs) for biological control of this pest. The four species of EPFs included Beauveria bassiana (strain GHA), Metarhizium brunneum (strain F52), Isaria javanica (wf GA17), and Isaria fumosorosea (Apopka 97 strain). In laboratory assays, all fungi reduced adult emergence but there were no differences between fungal species. Isaria javanica and M. brunneum were examined further in a EPFs and EPNs bioassay that also included the EPNs Steinernema carpocapsae (ALL strain) and S. riobrave (355 strain). All nematodes and fungi were applied either alone or in combination (fungus + nematode). There were no differences between species within the same entomopathogen group (fungi and nematodes). However, the treatment with S. riobrave resulted in lower R. pomonella emergence than either fungal species. The combination of S. riobrave and I. javanica resulted in the lowest R. pomonella emergence (3%) at fourth-week interval, which was significantly lower than any of the single-agent applications, yet virulence of the other three combination treatments was not different from their respective nematode treatments applied alone. Additive interactions were detected for all fungus–nematode combinations. This study suggests that application of entomopathogenic nematodes and fungi could be an effective option to suppress R. pomonella populations.

Objective and design: Our research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model. Methods C57Bl/6 or CD14-deficient mice were infected intratracheally with A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. Results CD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load control. The lungs of CD14-deficient mice presented a smaller area of ​​tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and sCD14 levels in BALF and serum of infected mice. Conclusions In summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.

Helminth infection currently affect over 2 billion people worldwide, with those with the most pathologies and morbidities, living in regions with unequal and disproportionate access to effective healthcare solutions. Host genetics and environmental factors play critical roles in modulating and regulating immune responses following exposure to various pathogens and insults. However, the interplay of environment and genetic factors in influencing who gets infected and the establishment, persistence, and clearance of helminth parasites remains unclear. Inbred strains of mice have long been used to investigate the role of host genetic factors on pathogenesis and resistance to helminth infection in a laboratory setting. This review will discuss the use of ecological and environmental mouse models to study helminth infections and how this could be used in combination with host genetic variation to explore the relative contribution of these factors in influencing immune response to helminth infections. Improved understanding of interactions between genetics and the environment to helminth immune responses would be important for efforts to identify and develop new prophylactic and therapeutic options for the management of helminth infections and their pathogenesis.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10–7 mol L−1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL−1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

Babesia is tick-transmitted protozoan parasites that infect mammalian hosts and have a major impact on farm and pet health-associated costs worldwide. This study aimed to test the prevalence of Babesia spp. infection in a small cohort of dogs at a veterinary hospital and to perform molecular characterization of the Babesia species causing the infection. For the PCR assay, 5 mL of blood was collected by venipuncture of the cephalic or radial veins in 300 dogs of different ages, sex, and breeds, which were presented to the veterinary hospital of the Federal University of Uberlândia between March 2015 and April 2016. In addition, a drop of blood was collected from the marginal blood vessels of the ear of dogs included in this study. Ninety-two (30.67%) were positive for Babesia spp., as determined by microscopic observation of the blood smear, revealing the presence of intra-erythrocyte merozoites. For molecular characterization by PCR, 17 samples were chosen from dogs who were tested positive for Babesia spp. by blood smears. Among them, B. vogeli was found to infect all 17 dogs, as determined by 99-100% sequence identity (closest GenBank match KT246307) using primers PIRO A/PIRO B. Our results indicate that the species observed in these dogs was B. vogeli.

Circadian rhythm is essential for cellular regulation of physiological, metabolic, and immune functions. Perturbations of circadian rhythms have been correlated with increased susceptibility to cancer and poor prognosis in the cancer treatment. Our aim is to investigate the role of doxorubicin (DOX) treatment on clock genes expression and inflammation in intraperitoneal macrophages and the antitumoral response. Methods: Macrophages were extracted from intraperitoneal cavity of mice without or with Lewis lung carcinoma (LLC) and treated with DOX totaling four groups (CTL, LLC, LLC+DOX and DOX) and analyzes of clock genes in six time points (ZT02, ZT06, ZT10, ZT14, ZT18 AND ZT22). Intraperitoneal macrophages cell culture was stimulated with LPS and DOX and clock genes and inflammatory profile were analyzed. In tumor were analyzed macrophages markers. Results: The expression of F4/80 (ZT22) and CD11c (ZT06) tumor tissue was significantly differed between LLC and LCC+DOX groups. In the intraperitoneal macrophages, DOX increased Clock (ZT10), Rev-Erbα (ZT18 and ZT22) and Per2 expressions (ZT18); in the LLC+DOX group was increased Bmal1 (ZT10), Per2 (ZT18) and NF-kB (ZT22) expressions; IL-6 expression increased in the LCC group (ZT02). In intraperitoneal macrophages cell culture stimulated with DOX and LPS after 24 h decreased Clock and Per1. DOX causes depression after 6 and 24 h in TNF-α content and Per2 gene expression after 24 h IL-1β expression was reduced also. Conclusion: DOX treatment in vivo disrupted cytokine and clock genes expression in intraperitoneal macrophages suppressing immune response. Moreover, macrophages cultured with DOX had decreased expression of LPS-stimulated inflammatory cytokines.

Made available in DSpace on 2020-12-12T02:24:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: The initiation and progression of periodontitis might involve a local renin-angiotensin system in periodontal tissue. This study hypothesized that Losartan treatment could promote protection to rats submitted to experimental periodontitis (EP) by attenuating alveolar bone loss due to reduction in inflammatory cytokines, better reactive oxidant species regulation and maintenance of the balance between bone formation and resorption factors. Methods: One hundred and thirty rats were submitted to EP with a silk suture thread (4.0) placed around the lower right first molar for 1, 3, 7, and 14 consecutive days. The study comprised four groups: G1—control without EP; G2—animals with EP treated with water; G3—Losartan-treated animals (treatment started at the same day of EP induction), and G4—animals previously treated with Losartan for 30 days followed by induction of EP and continuity of treatment. Results: G2 rats had greater bone loss volume, increased number, and thickness and decreased separation of trabeculae. On the other hand, G4 animals showed significant improvements in these parameters. Histological analysis revealed that EP favors inflammatory cell infiltration and junctional epithelium, cementum with alveolar bone crest destruction, but animals pretreated with Losartan (G4) did not show these features. Although the G3 animals did not demonstrate the improvements detected in G4, mRNA expression results were similar. In mandibular tissue, EP promoted mRNA increases for ACE, AT1 receptor, and inflammatory mediators as well as decreases for antioxidant enzymes. However, Losartan treatments attenuated these responses in addition to promoting an increase in bone formation markers and transcription factors. Conclusion: AT1 receptor modulates EP progression. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Department of Basic Sciences School of Dentistry São Paulo State University-UNESP Department of Biomedical Engineering Medical College of Wisconsin Department of Basic Sciences School of Dentistry São Paulo State University-UNESP FAPESP: 2015/03965-2

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Endothelial dysfunction and inflammation are recognised factors in the development of atherosclerosis. Evidence suggests that intake of industrial trans fatty acids (TFAs) promotes endothelial dysfunction, while ruminant TFAs may have the opposite effect. The aim of this study was to compare the effects of elaidic acid (EA (18:1n-9t), an industrially produced TFA) and trans vaccenic acid (TVA (18:1n-7t), a natural TFA found in ruminant milk and meat) on inflammatory responses of endothelial cells (ECs). ECs (EA.hy926 cells) were cultured under standard conditions and exposed to TFAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments. TFAs were incorporated into ECs in a dose-dependent manner. Preincubation with EA (50 µM) increased production of MCP-1, RANTES, and IL-8 in response to TNF-α, while preincubation with TVA (1 µM) decreased production of ICAM-1 and RANTES in response to TNF-α. Preincubation with EA (50 µM) upregulated toll-like receptor 4 and cyclooxygenase 2 gene expression in response to TNF-α. In contrast, preincubation with TVA (1 µM) downregulated TNF-α induced nuclear factor kappa B subunit 1 gene expression. Preincubation of ECs with EA (50 µM) increased THP-1 monocyte adhesion. In contrast, preincubation of ECs with TVA (1 µM) reduced THP-1 monocyte adhesion, while preincubation of ECs with TVA (50 µM) decreased the level of surface expression of ICAM-1 seen following TNF-α stimulation. The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. These findings suggest differential effects induced by the TFAs tested, fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions in an inflammatory context.

Seven transmembrane receptors (7TMRs), often termed G protein-coupled receptors (GPCRs), are the most common target of therapeutic drugs used today. Many studies suggest that distinct members of the GPCR superfamily represent potential targets for the treatment of various metabolic disorders including obesity and type 2 diabetes (T2D). GPCRs typically activate different classes of heterotrimeric G proteins, which can be subgrouped into four major functional types: Gαs, Gαi, Gαq/11, and G12/13, in response to agonist binding. Accumulating evidence suggests that GPCRs can also initiate β-arrestin-dependent, G protein-independent signaling. Thus, the physiological outcome of activating a certain GPCR in a particular tissue may also be modulated by β-arrestin-dependent, but G protein-independent signaling pathways. In this review, we will focus on the role of G protein- and β-arrestin-dependent signaling pathways in the development of obesity and T2D-related metabolic disorders.

A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.

Background Surface functionalization enhances the properties and characteristics of polymeric nanocapsules (NCs) mainly due to the surface charge, surfactants, and polymer coating type. Curcumin (CUR) is a bioactive compound with several proven pharmacological properties and low bioavailability. This study aimed to develop anionic (poly-ɛ-caprolactone; PCL) and cationic (Eudragit® RS100 (EUD)) NCs prepared with sorbitan monostearate (Span 60®) or sorbitan monooleate (Span 80®), coated with d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and optimized using 23 factorial analysis. Subsequently, the biological activity was evaluated. Methods A two-level, three-factor design (polymer, Span type, and TPGS concentration) was used. The biological effects of CUR-loaded TPGS-coated cationic and anionic NCs were assessed in apomorphine-induced stereotyped behavior in rats. Results The type of polymer (anionic or cationic) and Span® had a factorial influence on the physical and chemical characteristics of NCs according to the changes in TPGS concentrations. Both cationic and anionic CUR-NCs could block apomorphine-induced behavioral changes. Conclusions The CUR-loaded TPGS-coated NCs proved to be a promising brain delivery system.

Tobacco thrips, Frankliniella fusca (Hinds) is an economically significant pest. Entomopathogenic nematodes (EPNs) have shown promise as biocontrol agents against certain thrips species, but they have not been explored for suppression of F. fusca. We investigated the potential of EPNs to manage F. fusca by conducting three different bioassays: 1) a small cup dose–response bioassay (25, 50, and 100 IJs cm−2) with four EPN species, 2) a broad virulence bioassay with eight EPN species at 100 IJs cm−2, and 3) a potted soil bioassay testing with four EPN species (100 IJs cm−2). In the dose–response bioassay, all treatments showed relatively lower adult emergence when compared with the control group, but the minimum adult emergence (30%) was observed at 7 d post-treatment when Heterorhabditis bacteriophora (FL1-1) was applied at the highest rate (100 IJs cm−2). In the broad virulence study, all EPN treatments caused significant reductions in F. fusca adult emergence (18.3–75.0%) in comparison with the control. H. bacteriophora (Fl1-1) was more virulent than other nematode treatments but statistically not different from Steinernema feltiae and Steinernema riobrave, while Steinernema rarum was the least virulent. In the potted soil bioassay, the lowest emergence (10.6%) was observed in H. bacteriophora (Fl1-1) treatment, followed by S. feltiae (SN), S. riobrave (355), and Heterorhabditis indica (HOM1) treatments. These results indicate that EPNs have the ability to suppress the soil dwelling stage of F. fusca and should be explored further under greenhouse and field conditions for biocontrol potential within an integrated pest management (IPM) context.

Made available in DSpace on 2022-04-28T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Liver steatosis is one of the main drivers for the development of whole-body insulin resistance. Conversely, aerobic training (AT) has been suggested as non-pharmacological tool to improve liver steatosis, however, the underlying molecular mechanism remains unclear. Therefore, the aim of this study was to analyze the effect of 8-weeks AT in non-alcoholic liver disease (NAFLD) outcomes in obese mice. Male C57BL/6 J wild type (WT) were fed with standard (SD) or high-fat diet (HFD) for 12-weeks. Another group fed with HFD underwent 8-weeks of AT (60% of maximum velocity), initiated at the 5th week of experimental protocol. We measured metabolic, body composition parameters, protein and gene expression inflammatory and metabolic mediators. We found that AT attenuates the weight gain, but not body fat accumulation. AT improved triacylglycerol and non-esterified fatty acid plasma concentrations, and also whole-body insulin resistance. Regarding NAFLD, AT decreased the progression of macrovesicular steatosis and inflammation through the upregulation of AMPK Thr172 phosphorylation and PPAR-α protein expression. Moreover, although no effects of intervention in PPAR-γ protein concentration were observed, we found increased levels of its target genes Cd36 and Scd1 in exercised group, demonstrating augmented transcriptional activity. AT reduced liver cytokines concentrations, such as TNF-α, IL-10, MCP-1 and IL-6, regardless of increased Ser536 NF-κB phosphorylation. In fact, none of the interventions regulated NF-κB target genes Il1b and Cccl2, demonstrating its low transcriptional activity. Therefore, we conclude that AT attenuates the progression of liver macrovesicular steatosis and inflammation through AMPK-PPAR-α signaling and PPAR-γ activation, respectively, improving insulin resistance in obese mice. Immunometabolism Research Group Department of Cell and Developmental Biology University of São Paulo, Avenida Prof Lineu Prestes, 1524 Institute of Biomedical Sciences University of Sao Paulo Exercise and Immunometabolism Research Group Department of Physical Education University of the State of Sao Paulo, Rua Roberto Simonsen, 305 FAPESP: 2016/02696-0 FAPESP: 2019/09854-9

Background Despite advances in cancer diagnosis and therapeutics, the overall 5-year survival rate of oral squamous cell carcinoma (OSCC) remains low. Tumor formation, progression, recurrence, and chemo-resistance are associated with the presence of cancer stem cells (CSC) that show phenotypic heterogeneity, but how they influence tumor behavior remains poorly understood. We aimed to describe how two CSC phenotypes from an OSCC cell line, CD44High ESAHigh (Epi-CSC) and CD44High ESALow (EMT-CSC), behave in vitro and in vivo. Methods In vitro behavior of FACS-sorted Epi-CSC and EMT-CSC from OSCC cells was characterized by their ability to form colonies, migrate, proliferate, and to invade a solid matrix. In vivo experiments were conducted in immunodeficient (NOD/SCID) mice by orthotopic xenografting of FACS-sorted OSCC subpopulations. Results In vitro, the Epi-CSC phenotype was more proliferative and generated more holoclones than the EMT phenotype. On the other hand, EMT-CSC migrate and invaded more than Epi-CSC cells in 3D culture, suggesting the CSC phenotype affects tumor cell behavior. When inoculated orthotopically into the tongues of immunodeficient mice, both subpopulations generated OSCC, but EMT-CSC formed fewer and smaller tumors. Conclusions Our results suggest that while cells in the Epi-CSC form the subpopulation that enables tumor growth, the EMT-CSC are related to migration and invasion. Clinically, this may reflect the importance of Epi-CSC for tumorigenesis and of the EMT-CSC for metastasis and highlights that variation in the proportion of CSC phenotypes from patient to patient may be relevant to the design of individual treatment protocols.

The earliest interaction between macrophages and Paracoccidioides brasiliensis is particularly important in paracoccidioidomycosis (PCM) progression, and surface proteins play a central role in this process. The present study investigated the contribution of β2 integrin in P. brasiliensis-macrophage interaction and PCM progression. We infected β2-low expression (CD18low) and wild type (WT) mice with P. brasiliensis 18. Disease progression was evaluated for fungal burden, lung granulomatous lesions, nitrate levels, and serum antibody production. Besides, the in vitro capacity of macrophages to internalize and kill fungal yeasts was investigated. Our results revealed that CD18low mice infected with Pb18 survived during the time analyzed; their lungs showed fewer granulomas, a lower fungal load, lower levels of nitrate, and production of high levels of IgG1 in comparison to WT animals. Our results revealed that in vitro macrophages from CD18low mice slowly internalized yeast cells, showing a lower fungal burden compared to WT cells. The migration capacity of macrophages was compromised and showed a higher intensity in the lysosome signal when compared with WT mice. Our data suggest that β2 integrins play an important role in fungal survival inside macrophages, and once phagocytosed, the macrophage may serve as a protective environment for P. brasiliensis.

FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Blastocystis spp. e Enterocytozoon bieneusi são parasitos do trato gastrointestinal de grande variedade de animais incluindo o homem. Estão presente em países desenvolvidos e nos em desenvolvimento e possuem potencial zoonótico. O objetivo desse estudo foi determinar a positividade e realizar caracterização molecular dos subtipos de Blastocystis spp. e genótipos de E. bieneusi, à partir de amostras fecais de animais de produção procedentes de propriedades rurais da microrregião de Uberlândia, MG, Brasil. Foram coletadas fezes de 50 galinhas, 35 porcos, 11 cavalos e 57 bovinos em 14 propriedades rurais. O DNA foi submetido às reações de PCR e nested-PCR para amplificação de fragmentos do gene SSU rDNA de Blastocystis spp. e dos genes ITS de E. bieneusi. Do total das 153 amostras coletadas, 93 (60,8%) foram positivas para Blastocystis spp. e 25 (16,3%) para E. bieneusi. Blastocystis spp. foi identificado em todas as propriedades analisadas e E. bieneusi, em nove. Foi realizado sequenciamento de nova geração (SNG) nas amostras positivas para Blastocystis spp. com identificação de 13 subtipos previamente conhecidos (ST1, ST3 ao 7, ST10, ST14, ST21, ST23 ao 26) e um subtipo novo, destes, seis são zoonóticos (ST1, ST3 ao 7). As galinhas estavam infectadas pelos ST1, ST3 ao 7, ST10, ST14, ST15, ST21, ST23, ST25 e 26; os porcos por ST1, ST3 ao 5; o cavalo pelos subtipos ST10, ST21, ST23, ST5 e 26 e os bovinos por ST1, ST4, ST5, ST10, ST14, ST21, ST23 ao 26 e por um ST Novo. O sequenciamento das amostras positivas de E. bieneusi, identificou oito genótipos relatados anteriormente (D, EbpA, H, PigEb1, CS-1, Peru11, BEB8 e I), todos potencialmente zoonóticos. Os genótipos D, EbpA e H foram identificados em galinhas e porcos; PigEb1 e CS-1 em porcos; Peru11 em galinhas; BEB8 e I em bovinos. Infecções concomitantes entre Blastocystis spp. e E. bieneusi foram observadas em 23 amostras, sendo três em galinhas (13%), 16 em porcos (69,6%) e quatro em bovinos (17,4%). A identificação de subtipos e genótipos considerados zoonóticos, presentes nos animais dessa pesquisa, sugere a possibilidade de haver transmissão entre animais e o homem. Blastocystis spp. and Enterocytozoon bieneusi are obligate intracellular parasites of the gastrointestinal tract of humans and a wide variety of animals. They are present in developed and developing countries and have zoonotic potential. The aim of this research was to determine the positivity and to perform molecular characterization of respectively Blastocystis spp. and E. bieneusi subtypes and genotypes in fecal samples from production animals from rural properties of microregion of Uberlândia, MG, Brazil. Feces form 50 chickens, 35 pigs, 11 horses and 57 cattle were collected from 14 rural properties. The DNA was extracted directly from feces and submitted to PCR and nested-PCR analysis for amplification of fragments of the SSU rDNA of Blastocystis spp. and of the ITS gene of E. bieneusi. Out of 153 samples collected, 93 (60,8%) were positive for Blastocystis spp. and 25 (16,3%) for E. bieneusi. Blastocystis spp. was identified in all properties and E. bieneusi in nine. Next generation sequencing (NGS) was performed on positive samples for Blastocystis spp. with identification of 13 subtypes previously identified (ST1, ST3 to 7, ST10, ST14, ST21, ST23 to 26) plus a new subtype; six are known to be zoonotic (ST1, ST3 to 7). Chickens were infected by ST1, ST3 to 7, ST10, ST14, ST21, ST23, ST25 and 26; pigs by ST1, ST3 to 5; the horse by ST10, ST21, ST23, ST25 to 26 and cattle by ST1, ST4 and 5, ST10, ST14, ST21, ST23 to 26 and a novel ST. Sequencing of E. bieneusi identified eight previously reported genotypes (D, EbpA, H, PigEb1, CS-1, Peru11, BEB8 and I), all potentially zoonotic. Genotypes D, EbpA and H were identified in chickens and pigs; PigEb and CS-1 in pigs; Peru11 in chickens and BEB8 and I in cattle. Concomitant parasite infections were observed in 23 samples, three from chickens (13%), 16 from pigs (69,6%) and four from cattle (17,4%). The identification of subtypes and genotypes considered zoonotic, present in the animals of this study, suggests the possibility of transmission between animals and humans. Tese (Doutorado)

Obesity is a disease characterized by the exacerbated increase of adipose tissue. A possible way to decrease the harmful effects of excessive adipose tissue is to increase the thermogenesis process, to the greater energy expenditure generated by the increase in heat in the body. In adipose tissue, the thermogenesis process is the result of an increase in mitochondrial work, having as substrate H+ ions, and which is related to the increased activity of UCP1. Evidence shows that stress is responsible for increasing the greater induction of UCP1 expression via β-adrenergic receptors. It is known that physical exercise is an important implement for sympathetic stimulation promoting communication between norepinephrine/epinephrine with membrane receptors. Thus, the present study investigates the influence of short-term strength training (STST) on fatty acid composition, lipolysis, lipogenesis, and browning processes in the subcutaneous adipose tissue (sWAT) of obese mice. For this, Swiss mice were divided into three groups: lean control, obesity sedentary, and obese strength training (OBexT). Obese animals were fed a high-fat diet for 14 weeks. Trained obese animals were submitted to 7 days of strength exercise. It was demonstrated that STST sessions were able to reduce fasting glycemia. In the sWAT, the STST was able to decrease the levels of the long-chain fatty acids profile, saturated fatty acid, and palmitic fatty acid (C16:0). Moreover, it was showed that STST did not increase protein levels responsible for lipolysis, the ATGL, ABHD5, pPLIN1, and pHSL. On the other hand, the exercise protocol decreased the expression of the lipogenic enzyme SCD1. Finally, our study demonstrated that the STST increased browning process-related genes such as PGC-1α, PRDM16, and UCP1 in the sWAT. Interestingly, all these biomolecular mechanisms have been observed independently of changes in body weight. Therefore, it is concluded that short-term strength exercise can be an effective strategy to initiate morphological changes in sWAT.

Recently, regeneration of CNS tracts has been partially accomplished by strategies of intrinsic neuronal growth stimulation. However, restoration of function is dependent on proper myelination of regenerating axons. Previous work from our group (Goulart et al., 2018) has shown an increase in oligodendrocyte staining in the regenerating optic nerve, 2 weeks after crush, in animals that were submitted to conditional deletion of pten gene in retinal ganglion cells and intravitreal injection of zymosan + cAMP. Thus, in the present study we aimed to investigate the maturation of the oligodendroglial lineage and myelination during the regeneration of the optic nerve under the same conditions of our previous work. We showed that the combined treatment promoted an increase of myelinated fibers within the optic nerve, 12 weeks after lesion, as well as an increase in Sox10 positive cells. Early-OPCs, positive to A2B5, were also increased at 12 weeks, whereas O4 positive, late-OPCs, were increased from 2 until 12 weeks after crush. At 12 weeks after crush, the optic nerve of Regenerating group presented more CC1 positive oligodendrocytes and increased MRF positive myelinating oligodendrocytes, culminating in CTB traced regenerating axons superimposed to MBP staining, suggestive of myelination. Thus, our work showed that conditional deletion of pten gene in retinal ganglion cells and intravitreal inflammatory stimuli + cAMP stimulate full maturation of the olidodendroglial lineage, from OPC proliferation and differentiation to myelination of regenerating CNS axons.

Although convincing experimental evidence indicates that Na+/K+/Cl− cotransporter 1 (NKCC1) is involved in spinal nociceptive information processing and in the generation of hyperalgesia and allodynia in chronic pain states, the cellular distribution of NKCC1 in the superficial spinal dorsal horn is still poorly understood. Because this important piece of knowledge is missing, the effect of NKCC1 on pain processing is still open to conflicting interpretations. In this study, to provide the missing experimental data, we investigated the cellular distribution of NKCC1 in the superficial spinal dorsal horn by immunohistochemical methods. We demonstrated for the first time that almost all spinal axon terminals of peptidergic nociceptive primary afferents express NKCC1. In contrast, virtually all spinal axon terminals of nonpeptidergic nociceptive primary afferents were negative for NKCC1. Data on the colocalization of NKCC1 with axonal and glial markers indicated that it is almost exclusively expressed by axon terminals and glial cells in laminae I–IIo. In lamina IIi, however, we observed a strong immunostaining for NKCC1 also in the dendrites and cell bodies of PV-containing inhibitory neurons and a weak staining in PKCγ-containing excitatory neurons. Our results facilitate further thinking about the role of NKCC1 in spinal pain processing.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique β-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the β-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of β-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

Background Exercise is a powerful tool for prevention and treatment of many conditions related to the cardiovascular system and also chronic low-grade inflammation. Peroxisome proliferator-activated receptors γ (PPARγ) exerts an import role on the regulation of metabolic profile and subsequent inflammatory response, especially in macrophages. Purpose To investigate the effects of 8-week moderate-exercise training on metabolic and inflammatory parameters in mice with PPARγ deficiency in myeloid cells. Methods Twelve-week old mice bearing PPARγ deletion exclusively in myeloid cells (PPARγlox/lox Lys Cre -/+ , knockout [KO]) and littermate controls (PPARγlox/lox Lys Cre -/- , wild type [WT]) were submitted to 8-week exercise training (treadmill running at moderate intensity, 5 days/week). Animals were evaluated for food intake, glucose homeostasis, serum metabolites, adipose tissue and peritoneal macrophage inflammation, and basal and stimulated cytokine secretion. Results Exercise protocol did not improve glucose metabolism or adiponectin concentrations in serum of KO mice. Moreover, the absence of PPARγ in macrophages exacerbated the proinflammatory profile in sedentary mice. Peritoneal cultured cells had higher tumor necrosis factor-α (TNF-α) secretion in nonstimulated and lipopolysaccharide (LPS)-stimulated conditions and higher Toll-4 receptor (TLR4) gene expression under LPS stimulus. Trained mice showed reduced TNF-α content in adipose tissue independently of the genotype. M2 polarization ability was impaired in KO peritoneal macrophages after exercise training, while adipose tissue-associated macrophages did not present any effect by PPARγ ablation. Conclusion Overall, PPARγ seems necessary to maintain macrophages appropriate response to inflammatory stimulus and macrophage polarization, affecting also whole body lipid metabolism and adiponectin profile. Exercise training showed as an efficient mechanism to restore the immune response impaired by PPARγ deletion in macrophages.

The aims of this study were to investigate whether the number of antral follicles (AF) in the ovaries of Nelore cows is influenced with the developmental competence of oocytes to reach the blastocyst stage and to quantify the mRNA abundance of genes associated with folliculogenesis and oogenesis in granulosa and cumulus cells. A total of 168 cows were distributed into two experimental groups according to the number of AF, low (≤31) and high AF (≥92), which were determined based on the mean number of AF (61.14) ± SD (30.43). Granulosa and cumulus cells were used to assess the mRNA expression of 16 genes. Cumulus cells from cows with low AF had higher mRNA expression of genes involved in meiosis resumption (NPR-2, NPR-3) and cumulus cell expansion (FGF10), as well as a transcription factor involved in the regulation of oocyte maturation and cell proliferation (STAT3). Conversely, granulosa cells from females with high AF had higher expression of PGR and AMHR2a, which are involved in meiosis resumption and cumulus cell expansion. Cumulus-oocyte complexes (COCs) were collected from 356 cows with low and high AF populations to evaluate embryo development. Cleavage and blastocyst rates did not differ between the groups. In conclusion, our findings revealed that genes involved in folliculogenesis and oogenesis are differently expressed in cumulus and granulosa cells of cows having low and high numbers of AF. These molecular differences suggest that the regulation of oocyte maturation, meiotic resumption and cumulus expansion may be influenced by the number of AFs. However, the variations in gene expression were not associated with in vitro oocyte developmental competence to reach the blastocyst stage, which confirms that oocytes from Nelore cows with low and high numbers of AF are similarly able to mature, regulate the fertilization process and support pre-implantation embryo development.

Palmitoleic acid (POA, 16:1n-7) is a lipokine that has potential nutraceutical use to treat non-alcoholic fatty liver disease. We tested the effects of POA supplementation (daily oral gavage, 300 mg/Kg, 15 days) on murine liver inflammation induced by a high fat diet (HFD, 59% fat, 12 weeks). In HFD-fed mice, POA supplementation reduced serum insulin and improved insulin tolerance compared with oleic acid (OA, 300 mg/Kg). The livers of POA-treated mice exhibited less steatosis and inflammation than those of OA-treated mice with lower inflammatory cytokine levels and reduced toll-like receptor 4 protein content. The anti-inflammatory effects of POA in the liver were accompanied by a reduction in liver macrophages (LM, CD11c+; F4/80+; CD86+), an effect that could be triggered by peroxisome proliferator activated receptor (PPAR)-γ, a lipogenic transcription factor upregulated in livers of POA-treated mice. We also used HFD-fed mice with selective deletion of PPAR-γ in myeloid cells (PPAR-γ KOLyzCre+) to test whether the beneficial anti-inflammatory effects of POA are dependent on macrophages PPAR-γ. POA-mediated improvement of insulin tolerance was tightly dependent on myeloid PPAR-γ, while POA anti-inflammatory actions including the reduction in liver inflammatory cytokines were preserved in mice bearing myeloid cells deficient in PPAR-γ. This overlapped with increased CD206+ (M2a) cells and downregulation of CD86+ and CD11c+ liver macrophages. Moreover, POA supplementation increased hepatic AMPK activity and decreased expression of the fatty acid binding scavenger receptor, CD36. We conclude that POA controls liver inflammation triggered by fat accumulation through induction of M2a macrophages independently of myeloid cell PPAR-γ., Graphical abstract Unlabelled Image, Highlights • Palmitoleic acid (POA) supplementation reduced serum insulin and improved insulin tolerance; • Livers of POA-treated mice exhibited less steatosis and inflammation; • POA lowered the liver M1 macrophages population and the expression of inflammation-related immune-cell markers; • POA increased PPAR-γ, a transcription factor that regulates anti-inflammatory effects in macrophages; • However, POA reduced liver inflammation even in mice that lack PPAR-γ expression in myeloid cells; • POA controls liver inflammation through induction of M2a macrophages independently of PPAR-γ in myeloid cells.

The purpose of current study was to evaluate the effect of curcumin (CUR) loaded lipid-core nanocapsules (CUR-LNC) treatment on neuroinflammatory and behavioral alterations in a model of sickness behavior induced by lipopolysaccharide (LPS) in rats. Rats were treated with CUR-LNC and CUR daily for 14 days. After the last treatments, sickness behavior was induced with LPS. Sickness behavior LPS-induced was confirmed by behavioral tests, an increase in levels of proinflammatory cytokines, decrease in levels of IL-10, overexpression of IDO-1 and IDO-2. In conclusion, CUR-LNC treatment attenuated the neuroinflammatory and behavioral changes caused in sickness behavior model.

Age and antral follicle count (AFC) are related to fertility in cattle, but this information remains limited in mares.To verify the influence of age and AFC on the reproductive characteristics of mares, 15 Quarter Horse donors, with 5-15 antral follicles, ranging from three to 17 years old, healthy and in good nutritional status, were divided into groups with low AFC (≤9 follicles) or high AFC (≥10 follicles) and mares considered young (≤9 years) or old (≥10 years). Mares were submitted to ultrasonography to determine the dominant follicle diameter, follicular growth rate, degree of uterine oedema and embryonic recovery for a minimum of three oestrous cycles.AFC was higher (P=0.001) in young mares compared with old mares. An interaction (P=0.001) between AFC and age was observed with regard to follicular growth rate, being that mares with low AFC and are old showed the lowest follicular growth compared with those of low AFC and young, high AFC and old, and high AFC and young. Younger mares and those with high AFC exhibited higher degree of uterine oedema (P0.05) on the third day of oestrus compared with older mares and with low AFC (3 and 2, respectively). However, in both groups, the degree of oedema reduced by the time of ovulation.Follicular growth rate, degree of uterine oedema and the number of antral follicles are higher in young mares with high AFC. However, the rate of embryonic recovery is not influenced by donors' age or AFC.

Oral squamous cell carcinoma (OSCC) is the most prevalent neoplasia of oral cavity worldwide and prognosis remains unchanged in decades. Recently, different authors reported that head and neck squamous cell carcinomas have a subpopulation of tumor initiating cells that apparently correspond to cancer stem cells (CSC) and are also responsible for tumor growth and metastasis. The purpose of the present study was to investigate the microscopic and phenotypic characteristics of OSCC tumors induced after orthotopic xenoimplantation of SCC9WT cell line and CSC-enriched subpopulation isolated from SCC9 cell line based on high expression of the putative CSC marker CD44. Different numbers of FACS-sorted SCC9 CD44high and CD44low cells as well as SCC9WT (wild type) were transplanted into the tongue of BALB/C nude (NOD/SCID) mice to evaluate their tumorigenic potential. Sixty days post-induction, tumors were morphologically characterized and immunostained for CSC markers (CD44, Nanog and Bmi-1), epithelial-mesenchymal transition (Snail, Slug) and epithelial differentiating cell markers (cytokeratins 4, 13, 15, 17 and 19), as well as E-cadherin and β-catenin. The data presented here shows that SCC9 CD44high cells have higher ability to form tumors than SCC9 CD44low cells, even when significantly lower numbers of SCC9 CD44high cells were transplanted. Immunoassessment of tumors derived from SCC9 CD44high cells revealed high expression of cytokeratin CK19, β-catenin, E-cadherin and CD44, and negative or low expression of CK17, CK4, CK15, CK13, Nanog, Bmi-1, Snail and Slug. While tumors derived from SCC9WT showed high expression of CK17, CK19, CD44, Nanog, Bmi-1, Snail and Slug, and negative or low expression of CK4, CK15, CK13, β-catenin and E-cadherin. Thus, SCC9 CD44high cells were highly tumorigenic, capable of originating heterogeneous tumors and these tumors have a immunohistochemical profile different from those formed by the wild type cell line.

This study was conducted to characterize the daily profile of testosterone secretion and its mean concentrations in the four seasons as well as to evaluate the semen characteristics and testicular biometry of Mangalarga Marchador stallions throughout the year in a tropical region. Three stallions were submitted to semen collections and evaluation of testicular biometry every 14 days along a year. Blood samples were collected once at the middle of each season, in a 20-min interval during 24 hr in order to evaluate the testosterone secretion profiles among seasons. Testosterone concentrations along the day were higher at the beginning of the afternoon (from 12:00 to 15:00 hr), but a circadian secretion was not clearly observed. Mean testosterone concentrations did not differ among seasons (p > .05), but a pattern of secretion along the day showed variations with higher concentrations in the afternoon during the winter. Ejaculate volume was higher during summer; however, sperm motility decreased in summer and spring. Total sperm in ejaculate, sperm morphology and testicular biometry kept constant along the year showing no differences among the seasons. The results demonstrated that in a tropical region, reproductive aspects of stallions did not show a clearly defined seasonal variation, and months of autumn and winter were not unsuitable for reproduction of the males.

The objective of this study was to investigate the effect of cyclodextrin-loaded cholesterol conjugates addition to freezing extenders on plasma membrane viability of frozen-thawed spermatozoa of the Piau swine breed. Twenty semen samples were used from five males. The freezing extender was based on lactose-egg yolk extender, added to 2% glycerol, 3% dimethylacetamide. The addition of cyclodextrin-loaded cholesterol conjugates was performed after centrifugation, when semen was diluted with the cooling extender. Four groups were subjected to the following treatment: without addition (group 1); 1.5 mg of cyclodextrin-loaded cholesterol/120 × 10 6 sperm (group 2); 1.5 mg of cyclodextrin-loaded cholestanol/120 × 10 6 sperm (group 3); 1.5 mg of cyclodextrin-loaded desmosterol/120 × 10 6 sperm (group 4). To check post-thawing sperm quality sperm motility and sperm morphology evaluation were used. Additionally, to check sperm viability the hypoosmotic swelling test, supravital staining, and fluorescent assay were used. The mean values recorded for total sperm motility of semen immediately after thawing were 54.5 ± 5.8, 55.5 ± 5.3, 53.7 ± 6.7, and 52.5 ± 6.6% respectively for groups one to four, without difference between themselves (p > 0.05). Regarding fluorescent assay the results were 28.3 ± 13.2, 26.9 ± 12.2, 22.2 ± 11.4, and 32.0 ± 15.3% respectively for groups one to four, also without difference between groups (p > 0,05). Similarly, complementary tests for evaluating the integrity and functionality of the plasma membrane showed no difference between treatments (p > 0.05). In conclusion, use of cyclodextrin-loaded cholesterol conjugates added to the plasma membrane of sperm did not demonstrate any additive effect on increasing and/or maintaining sperm motility.

This study was designed to examine fetal and maternal toxicity of curcumin (CURC) loaded lipid-core nanocapsules (LNC) prepared with poly(ϵ-caprolactone) as a polymer, administered during the organogenesis period. Free CURC and CURC loaded-LNC (C-LNC) (2 mg/kg), blank LNC (B-LNC) and saline (CONTROL) were administered per oral route from the 7° to 13° gestational day (GD). Dams were evaluated daily for body weight gain, clinical signs, water and food intake. On 20° GD, dams were euthanized, organs were weighed and blood was collected for biochemical determinations. Fetal biometrics and external morphological anomalies were assessed. Also, were performed histopathological analysis of placenta and measurement of cytokines levels in placental and fetal liver tissues. All groups did not cause changes in dams during the pregnancy. Furthermore, treatments did not cause external morphological changes and delayed fetal development. Still, for histopathological analysis of placental tissue, treatments did not cause alterations in evaluated parameters. For cytokines levels, CURC and C-LNC caused a decrease in placental levels of TNF-α. Therefore, we have demonstrated that C-LNC did not cause toxicological effects (mother and fetus), in the same manner as pattern bioactive compound, proving to be a promising nutraceutical delivery system for maternal supplementation with CURC.

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

Objectives In this study, polymeric nanoparticles based on chitosan incorporating the antifungal miconazole nitrate were fabricated and testedin vivo using murine vulvovaginal candidiasis. Methods Nanoparticles prepared by the ionotropic gelation method presented 200 to 300 nm diameter and polydispersity indexes ranging from 0.2 to 0.4. The nanoparticles were prepared to incorporate 63.9 mg/mL of miconazole nitrate to be testedin vivo. Murine vulvovaginal candidiasis was standardized using estradiol valerate before the animals were challenged by Candida albicans. Results The treatment using chitosan nanoparticles within miconazole nitrate presented the same therapeutic efficacy as miconazole nitrate in a commercial cream formulation, however using the antifungal content about seven-fold lower. This increase in the miconazole nitrate’s therapeutic efficacy is may be due to the down-regulation of interleukin 10 (IL-10) expression. Conclusions Our data represent a proof of concept that can be exploited to achieve an alternative and promising therapy for the treatment of vulvovaginal candidiasis.

Colorectal cancer affects the large intestine, leading to loss of white adipose tissue (WAT) and alterations in adipokine secretion. Lower incidence of colorectal cancer is associated with increased fibre intake. Fructooligosaccharides (FOS) are fibres that increase production of butyrate by the intestinal microbiota. Tributyrin, a prodrug of butyric acid, exerts beneficial anti-inflammatory effects on colorectal cancer. Our aim was to characterise the effects of diets rich in FOS and tributyrin within the context of a colon carcinogenesis model, and characterise possible support of tumorigenesis by WAT. C57/BL6 male mice were divided into four groups: a control group (CT) fed with chow diet and three colon carcinogenesis-induced groups fed either with chow diet (CA), tributyrin-supplemented diet (BUT), or with FOS-supplemented diet. Colon carcinogenesis decreased adipose mass in subcutaneous, epididymal, and retroperitoneal tissues, while also reducing serum glucose and leptin concentrations. However, it did not alter the concentrations of adiponectin, interleukin (IL)-6, IL-10, and tumour necrosis factor alpha (TNF)-&alpha, in WAT. Additionally, the supplements did not revert the colon cancer affected parameters. The BUT group exhibited even higher glucose tolerance and levels of IL-6, VEGF, and TNF-&alpha, in WAT. To conclude our study, FOS and butyrate supplements were not beneficial. In addition, butyrate worsened adipose tissue inflammation.

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.

Paracoccidioidomycosis (PCM) is a systemic mycosis, widespread in Latin America. PCM is a granulomatous disease characterized by a polymorphism of lesions depending on the pathogen's virulence, the immune status of the host and its genetic susceptibility. The thermodimorphic fungus Paracoccidioides brasiliensis was considered the only etiologic agent of PCM, yet recent works have shown significant genetic diversity among different strains of P. brasiliensis. Therefore, it has been proposed for a new species within the Paracoccidioides genus, named Paracoccidioides lutzii. To better understand the fungus-host interactions elicited by strains Pb01 and Pb18 as key representatives of P. lutzii and P. brasiliensis, respectively, we carried out studies to investigate differences in morphology, induced immune response, virulence and pathology between these two Paracoccidioides species. Our results demonstrate distinct patterns of host-parasite interaction and pathology caused by Pb18 and Pb01. These results open up new fronts for NEW: clinical studies, which may result in significant consequences for the diagnosis and treatment of PCM. Considering that our results cannot be extended to all strains of both species, more studies about the virulence among Paracoccioides must be explored in the future.

Although dietary fatty acids can modulate metabolic and immune responses, the effects of palmitoleic acid (16:1n-7) remain unclear. Since this monounsaturated fatty acid is described as a lipokine, studies with cell culture and rodent models have suggested it enhances whole body insulin sensitivity, stimulates insulin secretion by β cells, increases hepatic fatty acid oxidation, improves the blood lipid profile, and alters macrophage differentiation. However, human studies report elevated blood levels of palmitoleic acid in people with obesity and metabolic syndrome. These findings might be reflection of the level or activity of stearoyl-CoA desaturase-1, which synthesizes palmitoleate and is enhanced in liver and adipose tissue of obese patients. The aim of this review is to describe the immune-metabolic effects of palmitoleic acid observed in cell culture, animal models, and humans to answer the question of whether palmitoleic acid is a plausible nonpharmacological strategy to prevent, control, or ameliorate chronic metabolic and inflammatory disorders. Despite the beneficial effects observed in cell culture and in animal studies, there are insufficient human intervention studies to fully understand the physiological effects of palmitoleic acid. Therefore, more human-based research is needed to identify whether palmitoleic acid meets the promising therapeutic potential suggested by the preclinical research.

Background/Aims: The main purpose of this study was to investigate the effects of celecoxib and ibuprofen, both non-steroidal anti-inflammatory drugs (NSAIDs), on the decreased gluconeogenesis observed in liver of Walker-256 tumor-bearing rats. Methods: Celecoxib and ibuprofen (both at 25 mg/Kg) were orally administered for 12 days, beginning on the same day when the rats were inoculated with Walker-256 tumor cells. Results: Celecoxib and ibuprofen treatment reversed the reduced production of glucose, pyruvate, lactate and urea from alanine as well as the reduced production of glucose from pyruvate and lactate in perfused liver from tumor-bearing rats. Besides, celecoxib and ibuprofen treatment restored the decreased ATP content, increased triacylglycerol levels and reduced mRNA expression of carnitine palmitoyl transferase 1 (CPT1), while ibuprofen treatment restored the reduced mRNA expression of peroxisome proliferator-activated receptor alpha (PPARα) in the liver of tumor-bearing rats. Both treatments tended to decrease TNFα, IL6 and IL10 in the liver of tumor-bearing rats. Finally, the treatment with celecoxib, but not with ibuprofen, reduced the growth of Walker-256 tumor. Conclusion: Celecoxib and ibuprofen restored the decreased gluconeogenesis in the liver of Walker-256 tumor-bearing rats. These effects did not involve changes in tumor growth and probably occurred by anti-inflammatory properties of these NSAIDs, which increased expression of genes associated with fatty acid oxidation (PPARα and CPT1) and consequently the ATP production, normalizing the energy status in the liver of tumor-bearing rats.

Made available in DSpace on 2018-12-11T16:51:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The study aimed to evaluate the metabolic and inflammatory effects of short-term treatments (10 days) with metformin (MET) on the NAFLD caused by a high-fat diet (HFD) in C57BL/6 mice. After the treatment, histological liver slices were obtained, hepatocytes and macrophages were extracted and cultured with phosphate buffered saline, LPS (2.5 µg/mL) and MET (1 µM) for 24 h. Cytokine levels were determined by ELISA. NAFLD caused by the HFD was partially reduced by MET. The lipid accumulation induced by the HFD was not associated with liver inflammation; however, MET seemed to promote pro-inflammatory effects in liver, since it increased hepatic concentration of IL-1β, TNF-α, IL-6, MCP-1 and IFN-γ. Similarly, MET increased the concentration of IL-1β, IL-6 in hepatocyte cultures. However, in macrophages culture, MET lowered levels of IL-1β, IL-6 and TNF-α stimulated by LPS. Overall, MET reduced liver NAFLD but promoted hepatocyte increase in pro-inflammatory cytokines, thus, leading to liver inflammation. Immunometabolism Research Group Department of Cell Biology and Development Institute of Biomedical Sciences University of São Paulo Exercise and Immunometabolism Research Group Department of Physical Education UNESP Department of Physiology and Biophysics Institute of Biomedical Sciences University of São Paulo Cruzeiro do Sul University Exercise and Immunometabolism Research Group Department of Physical Education UNESP FAPESP: 2015/16777-0 FAPESP: 2016/01409-8

International audience; Alveolar macrophages play a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Monocytes are recruited from blood during inflammation and then mature into alveolar macrophages. The aim of this study was to investigate the effect of cigarette smoke (CS) at different times in lung macrophages and monocytes from blood and bone marrow in mice. Male mice (C57BL/6, n = 45) were divided into groups: control, CS 5 days, CS 14 days and CS 30 days. Five days' CS exposure induced a pronounced influx of neutrophils and macrophages in the lung associated with increased levels of keratinocyte chemoattractant (KC), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and matrix metalloproteinase (MMP)-12. After 14 days of CS exposure, neutrophil recruitment and cytokine production were greatly reduced. Moreover, chronic CS exposure led to increased recruitment of macrophages (with high expression of CD206), transforming growth factor-β (TGF-β) production as well as no detection of TNF-α, interleukin (IL)-6 and KC. CS can also change the monocyte phenotype in the blood and bone marrow, with an increase in Ly6C(low) cells. These results show for the first time that CS can change not only macrophage polarization but also monocyte. These results suggest that continued recruitment of Ly6C(low) monocytes may help the distinct renewing macrophage M2 population required for COPD progression.

Summary Palmitoleic acid (PM, 16:1n-7) has anti-inflammatory properties that could be linked to higher expression of PPARα, an inhibitor of NFκB. Macrophages play a major role in the pathogenesis of chronic inflammation, however, the effects of PM on macrophages are underexplored. Thus, we aimed to investigate the effects of PM in activated macrophages as well the role of PPARα. Primary macrophages were isolated from C57BL/6 wild type (WT) and PPARα knockout (KO) mice, cultured under standard conditions and exposed to lipopolysaccharides LPS (2.5 μg/ml) and PM 600 μmol/L conjugated with albumin for 24 hours. The stimulation with LPS increased the production of interleukin (IL)-6 and IL-1β while PM decreased the production of IL-6 in WT macrophages. In KO macrophages, LPS increased the production of tumour necrosis factor (TNF)-α and IL-6 and PM decreased the production of TNFα. The expression of inflammatory markers such NFκB and IL1β were increased by LPS and decreased by PM in both WT and KO macrophages. PM reduced the expression of MyD88 and caspase-1 in KO macrophages, and the expression of TLR4 and HIF-1α in both WT and KO macrophages, although LPS had no effect. CD86, an inflammatory macrophage marker, was reduced by PM independently of genotype. PM increased PPARγ and reduced PPARβ gene expression in macrophages of both genotypes, and increased ACOX-1 expression in KO macrophages. In conclusion, PM promotes anti-inflammatory effects in macrophages exposed to LPS through inhibition of inflammasome pathway, which was independent of PPARα, PPARϒ and AMPK, thus the molecular mechanisms of anti-inflammatory response caused by PM is still unclear.

Ingested fluoride (F) is absorbed mainly in the small intestine, which is controlled by the Enteric Nervous System (ENS). Although important intestinal symptomatology has been described after excessive F exposure, there have been no studies reporting the effects of F on the ENS. In this study, the effects of chronic F exposure were evaluated on the duodenums of rats through proteomic and morphological analyses. Concentrations of 0, 10, or 50 ppm of F were applied to the drinking water for 30 days. Immunofluorescence techniques were performed in the myenteric plexus of the duodenum to detect HuC/D, neuronal nitric oxide (nNOS), vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP), and substance P (SP). The 50 ppm F group presented a significant decrease in the density of nNOS-IR neurons. Significant morphological alterations were also observed in HUC/D-IR and nNOS-IR neurons; VIP-IR, CGRP-IR, and SP-IR varicosities for both groups (10 and 50 ppm F). Proteomic analysis of the duodenum demonstrated alterations in the expression of several proteins, especially those related to important biological processes, such as protein polymerization, which helps to explain the downregulation of many proteins upon exposure to 50 ppm of F.

Background/Aims: The objective of the current work was to test the effect of metformin on the tumor growth in rats with metabolic syndrome. Methods: We obtained pre-diabetic hyperinsulinemic rats by neonatal treatment with monosodium L-glutamate (MSG), which were chronically treated every day, from weaning to 100 day old, with dose of metformin (250 mg/kg body weight). After the end of metformin treatment, the control and MSG rats, treated or untreated with metformin, were grafted with Walker 256 carcinoma cells. Tumor weight was evaluated 14 days after cancer cell inoculation. The blood insulin, glucose levels and glucose-induced insulin secretion were evaluated. Results: Chronic metformin treatment improved the glycemic homeostasis in pre-diabetic MSG-rats, glucose intolerance, tissue insulin resistance, hyperinsulinemia and decreased the fat tissue accretion. Meanwhile, the metformin treatment did not interfere with the glucose insulinotropic effect on isolated pancreatic islets. Chronic treatment with metformin was able to decrease the Walker 256 tumor weight by 37% in control and MSG rats. The data demonstrated that the anticancer effect of metformin is not related to its role in correcting metabolism imbalances, such as hyperinsulinemia. However, in morphological assay to apoptosis, metformin treatment increased programmed cell death. Conclusion: Metformin may have a direct effect on cancer growth, and it may programs the rat organism to attenuate the growth of Walker 256 carcinoma.

The aim of the present study was to compare the lipid profile in oocytes of indicus and 1/2 indicus × taurus cows with high and low antral follicle count (AFC)/oocyte yields. After an OPU procedure (D0), antral follicles ≥3 mm were counted by ultrasonography (D4, 19, 34, 49, 64), and cows were assigned to groups with either high AFC (≥30 follicles; indicus, NH group; 1/2 indicus × taurus, AH group) or low AFC (≤15 antral follicles; indicus, NL group; 1/2 indicus × taurus, AL group). The lipid profiles of the oocytes were determined by MALDI-MS. For GI, GII and GIII oocytes, the indicus samples tend to cluster separately from the 1/2 indicus × taurus samples. The lipid species [PC (P-38:5) + H](+) and/or [PC (P-36:2) + Na](+) , [PC (38:2) + H](+) , [PC (38:5) + Na](+) and [TAG (60:8) + NH(4) ](+) were more abundant in indicus (NH and NL groups) than 1/2 indicus × taurus. The higher lipid content in the indicus oocytes likely reflects differences in the rate of lipid metabolism and may contribute to oocyte competence and embryo development.

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

The purpose of this study was to investigate the effect of infliximab, an anti-tumor necrosis factor α (TNFα) monoclonal antibody, on the progression of cachexia and several metabolic parameters affected by the Walker-256 tumor in rats.Infliximab (0.5 mg/kg) was ip administered, twice a day, beginning at the day in which the Walker-256 tumor cells were inoculated. After 12 days of treatment, the tumor growth, some parameters of cachexia/anorexia, the blood levels of triacylglycerol, glucose, lactate and urea, the peripheral response to insulin and the hepatic glycolysis and gluconeogenesis were investigated. The peripheral response to insulin was evaluated by the insulin tolerance test and the glycolysis and gluconeogenesis in isolated perfused liver.The treatment with infliximab did not alter the growth of the Walker-256 tumor, but attenuated (p0.05) the reduction of body weight and prevented (p0.05) the loss of retroperitoneal adipose tissue induced by the tumor. Moreover, treatment with infliximab tended to minimize the loss of gastrocnemius muscle, the reduction in food intake, the peripheral response to insulin and the liver gluconeogenesis from alanine, as well as the increased blood triacylglycerol, caused by the tumor. In contrast, treatment with infliximab did not attenuate the reduction in hepatic glycolysis and glycemia, nor did it minimize the rise in blood levels of lactate and urea induced by the tumor.The treatment with infliximab ameliorated some changes associated with cachexia, such as the reduction of adipose tissue and body weight, suggesting that TNFα plays a significant role in mediating these changes induced by the tumor. In addition, infliximab tended to improve or had no effect on other metabolic parameters affected by the Walker-256 tumor, suggesting that other mediators or tumor-related events are involved in these disorders.

Peroxisome proliferator-activated receptors (PPARs) play a major role in metabolism and inflammatory control. Exercise can modulate PPAR expression in skeletal muscle, adipose tissue, and macrophages. Little is known about the effects of PPAR-α in metabolic profile and cytokine secretion after acute exercise in macrophages. In this context, the aim of this study was to understand the influence of PPAR-α on exercise-mediated immune metabolic parameters in peritoneal macrophages. Mice C57BL/6 (WT) and PPAR-α knockout (KO) were examined in non-exercising control (n = 4) or 24 hours after acute moderate exercise (n = 8). Metabolic parameters (glucose, non-esterified fatty acids, total cholesterol [TC], and triacylglycerol [TG]) were assessed in serum. Cytokine concentrations (IL-1β, IL-6, IL-10, TNF-α, and MCP-1) were measured from peritoneal macrophages cultured or not with LPS (2.5 μg/mL) and Rosiglitazone (1 μM). Exercised KO mice exhibited low glucose concentration and higher TC and TG in serum. At baseline, no difference in cytokine production between the genotypes was observed. However, IL-1β was significantly higher in KO mice after LPS stimulus. IL-6 and IL-1β had increased concentrations in KO compared with WT, even after exercise. MCP-1 was not restored in exercised KO LPS group. Rosiglitazone was not able to reduce proinflammatory cytokine production in KO mice at baseline level or associated with exercise. Acute exercise did not alter mRNA expression in WT mice. Conclusion: PPAR-α seems to be needed for metabolic glucose homeostasis and anti-inflammatory effect of acute exercise. Its absence may induce over-expression of pro-inflammatory cytokines in LPS stimulus. Moreover, moderate exercise or PPAR-γ agonist did not reverse this response.

Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.