[6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

BACKGROUND/AIMS: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). METHODS: The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). RESULTS: The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. CONCLUSION: The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

Systemic and local inflammatory response after implantation of biomaterial in critical bone injuries.

PURPOSE: To evaluate inflammatory response in critical bone injuries after implantation of the biomaterial composed of hydroxyapatite (HA)/poly (lactic-coglycolic acid) (PLGA)/BLEED. METHODS: Forty-eight male Wistar rats (280 ± 20 grams) were divided into two groups: control group (CG), in which the animals do not receive any type of treatment; and biomaterial group (BG), in which the animals received the HA/PLGA/BLEED scaffold. Critical bone injury was induced in the medial region of the skull calotte with the aid of a trephine drill 8 mm in diameter. The biomaterial was implanted in the form of 1.5-mm thick scaffolds. Serum and calotte were collected at one, three and seven days. RESULTS: Biomaterial had a significant effect on the morphological structure of the bone, accelerating osteoblast activation within three days, without causing exacerbated systemic inflammation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that BG induced upregulation of osteogenic genes such as runt-related transcription factor 2, and stimulated genes of inflammatory pathways such as tumor necrosis factor-α, on the first day without overexpressing genes related to bone matrix degradation, such as tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-9. CONCLUSIONS: The HA/PLGA/BLEED® association can be used as a bone graft to aid bone repair, as it is capable of modulating expression of important genes at this stage of the repair process.

Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling.

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.

Schistosomiasis mansoni and hydrographical conditions in São Carlos, São Paulo, Brazil.

BACKGROUND: In Brazil, schistosomiasis mansoni cases still occur, even in non-endemic areas. This study aimed to evaluate schistosomiasis mansoni cases and to delimit water collections investigated for infested planorbidae in São Carlos, São Paulo, Brazil. METHODS: A cross-sectional descriptive study and spatial analysis of schistosomiasis mansoni cases notified in the city from January 2005 to December 2017 was conducted. The study used geographical information system software to map residential and leisure exposures to water courses and bodies and related them to planorbidae surveys of São Paulo state. RESULTS: During the study period, 32 cases were notified. The main forms were intestinal and hepatosplenic. Twenty-eight cases were allochthonous, two autochthonous and two indeterminate. Eleven patients (33.3%) had contact with water collections in São Carlos, mainly the 29 and Broa reservoirs. Three of them had contact only with water collections in the region. A third of cases lived in the Água Fria and Água Quente microbasins, highly impacted by the presence of domestic sewage, and the whole region seems to be colonized by Biomphalaria tenagophila. CONCLUSIONS: The resolution of anthropogenic contamination of water bodies is crucial for controlling schistosomiasis mansoni autochthony in São Carlos.

Systemic and local inflammatory response after implantation of biomaterial in critical bone injuries

ABSTRACT Purpose: To evaluate inflammatory response in critical bone injuries after implantation of the biomaterial composed of hydroxyapatite (HA)/poly (lactic-coglycolic acid) (PLGA)/BLEED. Methods: Forty-eight male Wistar rats (280 ± 20 grams) were divided into two groups: control group (CG), in which the animals do not receive any type of treatment; and biomaterial group (BG), in which the animals received the HA/PLGA/BLEED scaffold. Critical bone injury was induced in the medial region of the skull calotte with the aid of a trephine drill 8 mm in diameter. The biomaterial was implanted in the form of 1.5-mm thick scaffolds. Serum and calotte were collected at one, three and seven days. Results: Biomaterial had a significant effect on the morphological structure of the bone, accelerating osteoblast activation within three days, without causing exacerbated systemic inflammation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that BG induced upregulation of osteogenic genes such as runt-related transcription factor 2, and stimulated genes of inflammatory pathways such as tumor necrosis factor-α, on the first day without overexpressing genes related to bone matrix degradation, such as tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-9. Conclusions: The HA/PLGA/BLEED® association can be used as a bone graft to aid bone repair, as it is capable of modulating expression of important genes at this stage of the repair process.

Perfil de IL-6 e TNF na Formação de Células Espumosas: Um Método Aprimorado Usando a Sonda de Isotiocianato de Fluoresceína (FITC) / Profile of IL-6 and TNF in Foam Cell Formation: An Improved Method Using Fluorescein Isothiocyanate (FITC) Probe

Resumo Fundamento A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose. Objetivo Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo. Métodos A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 μg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo. Resultados Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 μg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle. Conclusões Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.

Abstract Background The formation of foam cells occurs due to the increase in low-density plasma lipoprotein (LDL) and dysregulation of inflammation, which is important for the development of atherosclerosis. Objective To evaluate the profile of tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) in the existing foam cell formation method, optimizing this protocol. Methods The LDL was isolated, oxidized, and labeled with a Fluorescein isothiocyanate (FITC) probe. Foam cells were generated from THP-1 human monocyte-derived cells and incubated in the absence (control) or presence of FITC-ox-LDL (10, 50, 100, 150, or 200 μg/mL), for 12, 24, 48, or 72 hours. The accumulated FITC-ox-LDL in the cell was quantified by microscopy. The enzyme-linked immunosorbent assay was evaluated to quantify the IL-6 and TNF-α, with p < 0.05 considered significant. Results All the FITC-ox-LDL concentrations tested showed a higher fluorescence when compared to the control, showing a greater accumulation of lipoprotein in cells. The higher the concentration of FITC-ox-LDL, the greater the production of TNF-α and IL-6. The production of IL-6 by foam cells was detected up to the value of 150 µg/mL of the maximum stimulus for LDL. Concentrations above 50 μg/mL LDL stimulated greater release of TNF-α compared to control. Conclusions Our model contributes to the understanding of the release of IL-6 and TNF-α in response to different concentrations of ox-LDL, using an optimized method for the formation of foam cells.

Profile of IL-6 and TNF in Foam Cell Formation: An Improved Method Using Fluorescein Isothiocyanate (FITC) Probe. / Perfil de IL-6 e TNF na Formação de Células Espumosas: Um Método Aprimorado Usando a Sonda de Isotiocianato de Fluoresceína (FITC).

BACKGROUND: The formation of foam cells occurs due to the increase in low-density plasma lipoprotein (LDL) and dysregulation of inflammation, which is important for the development of atherosclerosis. OBJECTIVE: To evaluate the profile of tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) in the existing foam cell formation method, optimizing this protocol. METHODS: The LDL was isolated, oxidized, and labeled with a Fluorescein isothiocyanate (FITC) probe. Foam cells were generated from THP-1 human monocyte-derived cells and incubated in the absence (control) or presence of FITC-ox-LDL (10, 50, 100, 150, or 200 µg/mL), for 12, 24, 48, or 72 hours. The accumulated FITC-ox-LDL in the cell was quantified by microscopy. The enzyme-linked immunosorbent assay was evaluated to quantify the IL-6 and TNF-α, with p < 0.05 considered significant. RESULTS: All the FITC-ox-LDL concentrations tested showed a higher fluorescence when compared to the control, showing a greater accumulation of lipoprotein in cells. The higher the concentration of FITC-ox-LDL, the greater the production of TNF-α and IL-6. The production of IL-6 by foam cells was detected up to the value of 150 µg/mL of the maximum stimulus for LDL. Concentrations above 50 µg/mL LDL stimulated greater release of TNF-α compared to control. CONCLUSIONS: Our model contributes to the understanding of the release of IL-6 and TNF-α in response to different concentrations of ox-LDL, using an optimized method for the formation of foam cells.

FUNDAMENTO: A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose. OBJETIVO: Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo. MÉTODOS: A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 µg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo. RESULTADOS: Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 µg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle. CONCLUSÕES: Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.

Efficacy of instillation of MB49 cells and thermoreversible polymeric gel in urothelial bladder carcinoma immunization.

BACKGROUND: Activating the immune system for therapeutic benefit has long been a goal in immunology, especially in cancer treatment, but the low immunogenicity of antitumor vaccines remains a limiting factor in the fight against malignant neoplasms. The increase in the immunogenicity of weak antigens using biodegradable polymers, such as chitosan, has been observed in the field of cancer immunotherapy. However, the effects of the vaccine using a combination of tumor cells and a thermoreversible delivery system based on chitosan in bladder cancer models, mainly using the intravesical route to stimulate the antitumor immune response, are unknown. We propose to evaluate the efficacy of a polymeric gel matrix (TPG) formed by poloxamer 407 and chitosan, associated with MB49 cells, as an intravesical antitumor vaccine using a C57BL/6 murine model of bladder urothelial carcinoma. The effectiveness of immunization was analyzed with the formation of three experimental groups: Control, TPG and TPG + MB49. In the vaccination phase, the TPG + MB49 group underwent a traumatic injury to the bladder wall with immediate intravesical instillation of the vaccine compound containing MB49 cells embedded in TPG. The TPG group was subjected to the same procedures using the compound containing the gel diluted in medium, and the control group using only the medium. After 21 days, the animals were challenged with tumor induction. RESULTS: In vitro tests showed loss of viability and inability to proliferate after exposure to TPG. In vivo tests showed that animals previously immunized with TPG + MB49 had higher cumulative survival, as well as significantly lower bladder weight and size in contrast to the other two groups that did not show a statistically different tumor evolution. In addition, the splenocytes of these animals also showed a higher rate of antitumor cytotoxicity in relation to the TPG and control groups. CONCLUSIONS: We can conclude that MB49 cells embedded in a polymeric thermoreversible gel matrix with chitosan used in the form of an intravesical vaccine are able to stimulate the immune response and affect the development of the bladder tumor in an orthotopic and syngeneic C57BL/6 murine model.

Schiff bases complexed with iron and their relation with the life cycle and infection by Schistosoma mansoni.

Introduction: The trematode Schistosoma mansoni causes schistosomiasis, and this parasite's life cycle depends on the mollusk Biomphalaria glabrata. The most effective treatment for infected people is administering a single dose of Praziquantel. However, there are naturally resistant to treatment. This work has developed, considering this parasite's complex life cycle. Methods: The synthetics compound were evaluated: i) during the infection of B. glabrata, ii) during the infection of BALB/c mice, and iii) during the treatment of mice infected with S. mansoni. Results and Discussion: For the first objective, snails infected with miracidia treated with compounds C1 and C3 at concentrations of 25% IC50 and 50% IC50, after 80 days of infection, released fewer cercariae than the infected group without treatment. For the second objective, compounds C1 and C3 did not show significant results in the infected group without treatment. For the third objective, the mice treated with C3 and C1 reduced the global and differential cell count. The results suggest that although the evaluated compounds do not present schistosomicidal properties when placed in cercariae suspension, they can stimulate an immune reaction in snails and decrease mice's inflammatory response. In general, we can conclude that compound C1 and C3 has an anti-schistosomicidal effect both in the larval phase (miracidia) and in the adult form of the parasite.

Elevations in Liver Transaminases in COVID-19: (How) Are They Related?

Liver involvement in COVID-19 is not yet well-understood, but elevations in liver transaminases have been described to occur in 14-53% of the cases and are more frequently seen in severe disease. This cross-sectional study explored the relationship between the elevations in liver transaminases and inflammatory parameters in 209 adults with COVID-19. Demographic and clinical data, serum levels of inflammatory cytokines and liver aminotransferases were analyzed. Three groups were formed according to the liver transaminase abnormalities: (I) Normal transaminases, (II) Borderline transaminases elevation, and (III) Mild to severe transaminases elevation. Altered liver transaminases were directly related to disease severity, showing association with the NEWS2 score at admission and greater need for ICU or death. Moreover, higher levels of IL-2 and CRP were associated with borderline transaminases elevations, whereas higher levels of IL-10 and Neutrophil to Lymphocyte ratio were associated with mild to severe transaminases elevation. These results reinforce the importance of liver transaminases in patients with COVID-19 as a complementary marker for disease severity and also point to them as a parameter reflecting the continuous dynamics between viral infection and the immune response.

Comparison of two different biomaterials in the bone regeneration (15, 30 and 60 days) of critical defects in rats.

PURPOSE: To evaluate and compare two types of different scaffolds in critical bone defects in rats. METHODS: Seventy male Wistar rats (280 ± 20 grams) divided into three groups: control group (CG), untreated animals; biomaterial group 1 (BG1), animals that received the scaffold implanted hydroxyapatite (HA)/poly(lactic-co-glycolic) acid (PLGA); and biomaterial group 2 (BG2), animals that received the scaffolds HA/PLGA/Bleed. The critical bone defect was induced in the medial region of the skull calotte with the aid of an 8-mm-diameter trephine drill. The biomaterial was implanted in the form of 1.5 mm thick scaffolds, and samples were collected after 15, 30 and 60 days. Non-parametric Mann-Whitney test was used, with the significance level of 5% (p ≤ 0.05). RESULTS: Histology revealed morphological and structural differences of the neoformed tissue between the experimental groups. Collagen-1 (Col-1) findings are consistent with the histological ones, in which BG2 presented the highest amount of fibers in its tissue matrix in all evaluated periods. In contrast, the results of receptor activator of nuclear factor kappa-Β ligand (Rank-L) immunoexpression were higher in BG2 in the periods of 30 and 60 days, indicating an increase of the degradation of the biomaterial and the remodeling activity of the bone. CONCLUSIONS: The properties of the HA/PLGA/Bleed scaffold were superior when compared to the scaffold composed only by HA/PLGA.

IL-6 and IL-10 are associated with disease severity and higher comorbidity in adults with COVID-19.

AIM: COVID-19 pandemic has caused extensive burden on public life and health care worldwide. This study aimed to assess circulating levels of inflammatory cytokines in adult patients who were hospitalized with COVID-19 and stratified according to age (older or younger than 65 years) aiming to explore associations between these markers of inflammation and comorbidities. METHODS: This was a cross-sectional study of 142 COVID-19 patients consecutively admitted to the University Hospital of the Federal University of São Carlos, from July to October 2020. Sociodemographic data, chronic comorbidities, and baseline NEWS2 and SOFA for clinical deterioration were obtained at hospital admission. Serum levels of inflammatory cytokines were determined by flow cytometry. RESULTS: Older adults with COVID-19 had higher serum levels of IL-6 and IL-10 as compared to those under 65 years of age (p < 0.001 and p = 0.003, respectively). IL-10 was independently associated with age (p = 0.04) and severity of the disease (p = 0.05), whereas serum levels of IL-6 were not directly associated with age (p = 0.5). The comorbidity index seems to be the main responsible for this, being significantly associated with IL-6 levels among those aged 65 and over (p = 0.007), in addition to the severity of the disease. CONCLUSIONS: Higher serum levels of IL-6 and IL-10 are associated with the severity of the disease and a higher comorbidity index among adults aged 65 and over with COVID-19. This should raise awareness of the importance of comorbidity index, rather than age, during risk stratification.

High-Intensity Interval Training Does Not Change Vaspin and Omentin and Does Not Reduce Visceral Adipose Tissue in Obese Rats.

This study aimed to determine the expression of omentin and vaspin, inflammatory markers, body composition, and lipid profile in diet-induced obese rats and high-intensity interval training (HIIT). Forty Wistar rats were divided into four groups: untrained normal diet, trained normal diet (T-ND), untrained high-fat diet (Unt-HFD), and trained high-fat diet (T-HFD). For the animals of the Unt-HFD and T-HFD groups, a high-fat diet was offered for 4 weeks. After that, all the animals in the T-ND and T-HFD groups were submitted to HITT, three times per week, for 10 weeks (2 weeks of adaptation and 8 weeks of HIIT). Muscle (gastrocnemius), liver, epididymal adipose tissue, retroperitoneal adipose tissue, visceral adipose tissue (VAT), and serum were collected to analyze TNF-α, IL-6, PCR, IL-8, IL-10, IL-4, vaspin, and omentin. A body composition analysis was performed before adaptation to HIIT protocol and after the last exercise session using dual-energy X-ray absorptiometry. Omentin and vaspin in the VAT were quantified using Western blotting. The results showed that, when fed a high-fat diet, the animals obtained significant gains in body fat and elevated serum concentrations of vaspin and blood triglycerides. The HIIT was able to minimize body fat gain but did not reduce visceral fat despite the increase in maximum exercise capacity. Moreover, there was a reduction in the serum levels of adiponectin, IL-6, and IL-10. Finally, we concluded that, although the training protocol was able to slow down the weight gain of the animals, there was no reduction in visceral fat or an improvement in the inflammatory profile, including no changes in omentin and vaspin.

Discovery of a Novel Lineage Burkholderia cepacia ST 1870 Endophytically Isolated from Medicinal Polygala paniculata Which Shows Potent In Vitro Antileishmanial and Antimicrobial Effects.

In this study, we report the isolation and identification of an endophytic strain of Burkholderia cepacia (COPS strain) associated with Polygala paniculata roots. Polygala plants are rich sources of promising microbiomes, of which the literature reports several pharmacological effects, such as trypanocidal, antinociceptive, anesthetic, anxiolytics, and anticonvulsant activities. B. cepacia COPS belongs to a new sequence type (ST 1870) and harbors a genome estimated in 8.3 Mbp which exhibits the aminoglycosides and beta-lactams resistance genes aph(3')-IIa and bla TEM-116, respectively. Analysis performed using MLST, average nucleotide identity, and digital DNA-DNA hybridization support its species-level identification and reveals its novel housekeeping genes alleles gyrB, lepA, and phaC. The root endophyte B. cepacia COPS drew our attention from a group of 14 bacterial isolates during the primary screening for being potentially active against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 9341, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 and exhibited the broad-spectrum activity against phytopathogenic fungi. In addition, COPS strain showed production of protease, lipase, and esterase in solid media, and its natural product extract showed potent inhibition against fungal plant pathogens, such as Moniliophthora perniciosa, whose antagonism index (89.32%) exceeded the positive control (74.17%), whereas Sclerotinia sclerotiorum and Ceratocystis paradoxa showed high percentages of inhibition (85.53% and 82.69%, respectively). COPS crude extract also significantly inhibited S. epidermidis ATCC 35984, E. faecium ATCC 700221 (MIC values of 32 µg/mL for both), E. faecalis ATCC 29212 (64 µg/mL), and S. aureus ATCC 25923 (128 µg/mL). We observed moderate antagonistic activity against A. baumannii ATCC 19606 and E. coli ATCC 25922 (both at 512 µg/mL), as well as potent cytotoxic effects on Leishmania infantum and Leishmania major promastigote forms with 78.25% and 57.30% inhibition. In conclusion, this study presents for the first time the isolation of an endophytic B. cepacia strain associated with P. paniculata and enough evidence that these plants may be considered a rich source of microbes for the fight against neglected diseases.

Comparison of two different biomaterials in the bone regeneration (15, 30 and 60 days) of critical defects in rats

ABSTRACT Purpose To evaluate and compare two types of different scaffolds in critical bone defects in rats. Methods Seventy male Wistar rats (280 ± 20 grams) divided into three groups: control group (CG), untreated animals; biomaterial group 1 (BG1), animals that received the scaffold implanted hydroxyapatite (HA)/poly(lactic-co-glycolic) acid (PLGA); and biomaterial group 2 (BG2), animals that received the scaffolds HA/PLGA/Bleed. The critical bone defect was induced in the medial region of the skull calotte with the aid of an 8-mm-diameter trephine drill. The biomaterial was implanted in the form of 1.5 mm thick scaffolds, and samples were collected after 15, 30 and 60 days. Non-parametric Mann-Whitney test was used, with the significance level of 5% (p ≤ 0.05). Results Histology revealed morphological and structural differences of the neoformed tissue between the experimental groups. Collagen-1 (Col-1) findings are consistent with the histological ones, in which BG2 presented the highest amount of fibers in its tissue matrix in all evaluated periods. In contrast, the results of receptor activator of nuclear factor kappa-Β ligand (Rank-L) immunoexpression were higher in BG2 in the periods of 30 and 60 days, indicating an increase of the degradation of the biomaterial and the remodeling activity of the bone. Conclusions The properties of the HA/PLGA/Bleed scaffold were superior when compared to the scaffold composed only by HA/PLGA.

Investigating Immunization With Nucleotide Enzymes of Schistosoma mansoni: Nucleoside Diphosphate Kinase and Adenylosuccinate Lyase as New Antigenic Targets Against Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.

In Vitro Modulator Effect of Total Extract from the Endophytic Paenibacillus polymyxa RNC-D in Leishmania (Leishmania) amazonensis and Macrophages.

Leishmaniases are diseases with high epidemiological relevance and wide geographical distribution. In Brazil, Leishmania (Leishmania) amazonensis is related to the tegumentary form of leishmaniasis. The treatment for those diseases is problematic as the available drugs promote adverse effects in patients. Therefore, it is important to find new therapeutic targets. In this regard, one alternative is the study of biomolecules produced by endophytic microorganisms. In this study, the total extract produced by the endophytic Paenibacillus polymyxa RNC-D was used to evaluate the leishmanicidal, nitric oxide, and cytokines production using RAW 264.7 macrophages. The results showed that, in the leishmanicidal assay with L. amazonensis, EC50 values at the periods of 24 and 48 hours were 0.624 mg/mL and 0.547 mg/mL, respectively. Furthermore, the cells treated with the extract presented approximately 25% of infected cells with an average of 3 amastigotes/cell in the periods of 24 and 48 hours. Regarding the production of cytokines in RAW 264.7 macrophages infected/treated with the extract, a significant increase in TNF-α was observed at the periods of 24 and 48 hours in the treated cells. The concentrations of IFN-γ and IL-12 showed significant increase in 48 hours. A significant decrease in IL-4 was observed in all cells treated with the extract in 24 hours. It was observed in the treated cells that the NO production by RAW 264.7 macrophages increased between 48 and 72 hours. The endophytic Paenibacillus polymyxa RNC-D extract modulates the mediators of inflammation produced by RAW 264.7 macrophages promoting L. amazonensis death. The immunomodulatory effects might be a promising target to develop new immunotherapeutic and antileishmanial drugs.

Biphasic Dose/Response of Photobiomodulation Therapy on Culture of Human Fibroblasts.

Objective: The objective of this study was to evaluate the effects of application of different fluences and energies of laser in the 24-, 48-, and 72-h periods in fibroblasts originating from human skin (HFF-1). Methods: The cell used as a template for cell proliferation was HFF-1. For the photobiomodulation (PBM) application, a 660 nm laser with a power of 40 mW and energies of 0.84, 1.40, 5.88, and 6.72 J was used. Five experimental groups were studied: one control group (CG) with simulated PBM and four groups that received PBM in different doses. The changes observed after laser irradiation were evaluated by cell viability (trypan blue) and proliferation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] tests. Intergroup comparisons were performed using two-way analysis of variance and the Tukey post hoc test (software GraphPad Prism 7.0). Results: In the trypan blue test, the total number of cells was significantly different between the irradiated groups and the CG at all times studied. The total number of cells increased in laser group (LG)1 (0.84 J) and LG2 (1.40 J) and decreased in LG4 (6.72 J). The mitochondrial activity increased significantly in LG1 and LG2 at 48 and 72 h and decreased in LG3 (5.88 J) and LG4 (6.72 J) compared with CG. Conclusions: The results indicate that the lower doses (0.45 and 0.75 J/cm2) of PBM induce the highest mitochondrial activity and cellular viability.

Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection.

Eosinophils are multifunctional cells that have cytotoxic proinflammatory activities and stimulate CD4+ T-cells in experimental models of allergy and parasitic infections. Eosinophils, when exposed to antigens, are activated, expressing the CD38/CD69 molecules and exhibited increased expression of major histocompatibility complex (MHC-II), CD80 and CD86, suggesting they play a role upon Toxocara canis antigen stimulation. In the present study, we evaluated the profile of eosinophils using conventional and image flow cytometry upon experimental T. canis infection. T. canis antigens induced a robust activation on this subset, contributing to the immune responses elicited in the experimental model for T. canis-associated visceral larva migrans syndrome. Data analysis demonstrated that, during murine T. canis infection, eosinophils from peripheral blood, spleen, and bone marrow presented upregulated expression of CD69/MHC-II/CD80/CD86. As opposed to splenic and bone marrow eosinophils, circulating eosinophils had increased expression of activation markers upon T. canis infection. The enhanced connectivity between eosinophils and T-cells in T. canis-infected mice in all three compartments (peripheral blood, spleen, and bone marrow) also supports the hypothesis that eosinophils may adopt a role during T. canis infection. Moreover, in vitro T. canis antigen stimulation resulted in activation and upregulation of co-stimulatory-related molecules by bone marrow-derived eosinophils. Our findings are evidence of activation and upregulation of important activation and co-stimulatory-related molecules in eosinophils and suggest a reshape of activation hierarchy toward eosinophils during experimental T. canis infection.