Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Carotenóides totais em Pothomorphe umbellata (L.) Miq. cultivadas in vitro

The aim of the present work was to compare the content of carotenoids between callus and regenerated plants of Pothomorphe umbellate. Germinated seeds (40 days old) were inoculated in different concentrations and combinations of BAP (benzylaminopurine) and NAA (naphthalene acetic acid) in order to stimulate the callus' production. After 60 days of culture, the callus containing some shoots were transferred to organogenesis medium (GA 3 0.1 mg L -1, BAP 0.5 mg L -1) for 40 days. Next, they were subcultivated in a medium for seedling growth (without regulators) for 40 days. Callus (collected after 60 days) and seedlings (collected after 140 days) were frozen in liquid nitrogen and kept under -80°C for future carotenoids' analysis. The highest concentration of carotenoids was found in plants cultivated in medium without regulators. The callus did not showed difference concerning the culture medium; however, they presented lower content of carotenoids in relation to plants.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes. © 2012 Springer-Verlag London Ltd.

High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro

Background: Dogs are commonly affected by hyperglycemic conditions. Hyperglycemia compromises the immune response and favors bacterial infections; however, reports on the effects of glucose on neutrophil oxidative metabolism and apoptosis are conflicting in humans and rare in dogs. Considering the many complex factors that affect neutrophil oxidative metabolism in vivo, we investigated in vitro the specific effect of high concentrations of glucose on superoxide production and apoptosis rate in neutrophils from healthy dogs.Results: The capacity of the neutrophils to reduce tetrazolium nitroblue decreased significantly in the higher concentration of glucose (15.13 ± 9.73% (8 mmol/L) versus 8.93 ± 5.71% (16 mmol/L)). However, there were no changes in tetrazolium nitroblue reduction at different glucose concentrations when the neutrophils were first activated with phorbol myristate acetate. High concentrations of glucose did not affect the viability and apoptosis rate of canine neutrophils either with or without prior camptothecin stimulation. This study provides the first evidence that high concentrations of glucose inhibit the oxidative metabolism of canine neutrophils in vitro in a manner similar to that which occurs in humans, and that the decrease in superoxide production did not increase the apoptosis rate.Conclusions: A high concentration of glucose reduces the oxidative metabolism of canine neutrophils in vitro. It is likely that glucose at high concentrations rapidly affects membrane receptors responsible for the activation of NADPH oxidase in neutrophils; therefore, the nonspecific immune response can be compromised in dogs with acute and chronic hyperglycemic conditions. © 2013 Bosco et al.; licensee BioMed Central Ltd.

Concentrações de sacarose no desenvolvimento in vitro e na aclimatização de Cattleya loddigesii Lindley

This work aimed to study the influence of sucrose in the culture medium for in vitro growth and acclimatization of the epiphytic orchid Cattleya loddigesii. Five sucrose treatments (absence, 10, 20, 30 and 40g L-1) were used in a randomic experimental design. Mature seeds were sowed in 1/2 MS culture medium and after 90 days the plantlets (1.0 +/-0.2 cm) were inoculated between the treatments, whereby they were remained more 90 days. After 180 days of the beginning of the experiment the plantlets were removed from the flasks and evaluated the number of roots, shoot length, number of leafs, total dry weight and photosynthetic pigments. Survival percentage was evaluated after 75 days of acclimatization. The data of biometric variables were analyzed by Anovaand polynomial regression (p<0.05). Theothers data were submitted to the Anova and the means compared by the Tukey test (p<0.05). The sucrose concentration of 20g L-1 favored the in vitro growth in all evaluated parameters, showed higher production of chlorophyll a, total chlorophyll and carotenoids, in addition to increased survival under ex vitro condition. The sucrose concentration of 20g L-1 in the culture medium was the most efficient among the tested concentrations both for in vitro growth and ex vitro establishment of Cattleya loddigesii.

Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (106 cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 μM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log 10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL -1 for S. mutans biofilms (p = 0.001), and 0.95 and 0.88 log 10 CFU mL-1 for S. sanguinis biofilms (p = 0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases. © 2012 Springer-Verlag London Ltd.

In vitro transdentinal effect of low-level laser therapy

Low-level laser therapy (LLLT) has been used for the treatment of dentinal hypersensitivity. However, the specific LLL dose and the response mechanisms of these cells to transdentinal irradiation have not yet been demonstrated. Therefore, this study evaluated the transdentinal effects of different LLL doses on stressed odontoblast-like pulp cells MDPC-23 seeded onto the pulpal side of dentin discs obtained from human third molars. The discs were placed in devices simulating in vitro pulp chambers and the whole set was placed in 24-well plates containing plain culture medium (DMEM). After 24 h incubation, the culture medium was replaced by fresh DMEM supplemented with either 5% (simulating a nutritional stress condition) or 10% fetal bovine serum (FBS). The cells were irradiated with doses of 15 and 25 J cm-2 every 24 h, totaling three applications over three consecutive days. The cells in the control groups were removed from the incubator for the same times as used in their respective experimental groups for irradiation, though without activating the laser source (sham irradiation). After 72 h of the last active or sham irradiation, the cells were evaluated with respect to succinic dehydrogenase (SDH) enzyme production (MTT assay), total protein (TP) expression, alkaline phosphatase (ALP) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) for collagen type 1 (Col-I) and ALP, and morphology (SEM). For both tests, significantly higher values were obtained for the 25 J cm-2 dose. Regarding SDH production, supplementation of the culture medium with 5% FBS provided better results. For TP and ALP expression, the 25 J cm-2 presented higher values, especially for the 5% FBS concentration (Mann-Whitney p < 0.05). Under the tested conditions, near infrared laser irradiation at 25 J cm -2 caused transdentinal biostimulation of odontoblast-like MDPC-23 cells. © 2013 Astro Ltd.

Pulp fruit added to culture medium for in vitro orchid development

As an additive in in vitro culture media, fruits have a great potential for facilitating economical orchid production because of lower technology requirements and the ease of obtaining raw materials to formulate culture media. We studied the in vitro growth of Cattleya bicolor Lindl. grown in a simplified culture medium supplemented with different kinds of fruit pulp. The experimental design was completely randomised, with eight seedlings per replication and ten replications per treatment, for a total of 80 seedlings per treatment. The culture medium was made using 150 g L -1 of pulp (without peel or seed) from the following fruits: ripe Santa Cruz tomatoes (Solanum lycopersicum L.), dwarf bananas (Musa cavendishii L.) of intermediate ripeness, light green chayote (Sechium edule (Jacq.) Sw), ripe papaya (Carica papaya L.) or green coconut (Cocos nucifera L.).The treatment control was MS 50 %. The treatments and the control were kept in a growth chamber for seven months before evaluating seedling survival percentage, shoot height, number of leaves, rooting percentage, root number, root length and dry masses of shoot and roots. The highest percentages of seedling survival were obtained using MS 50 %, banana and coconut medium. The seedling survival and rooting percentages illustrate that it is possible to emphasise the culture medium MS 50% and the culture medium supplemented with coconut on the most traditional culture medium with banana or tomato pulp. For the in vitro development of Cattleya bicolor Lindl., a simplified culture medium supplemented with coconut pulp is the most suitable for use as an alternative to MS 50%. A simplified culture medium supplemented with papaya pulp is not recommended for the in vitro development of Cattleya bicolor Lindl.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Atividade antimicrobiana in vitro de extrato de Punica granatum L. sobre staphylococcus aureus isolado em leite bovino

Bovine mastitis is considered important disease causing major economic losses in dairy herds. Antimicrobial drug can promote resistance, chemical residues in food and environmental contamination. The purpose of this study was to evaluate the antibacterial activity in vitro of pomegranate extract on Staphylococcus aureus isolated from bovine milk, evaluate its antioxidant activity, and quantify levels of total phenols and fravonoides of the different extracts used. Aqueous extracts were used in nature and dry, from the peel of the fruit (EAC) and leaves (EAF). Additionally, it was evaluated the antioxidant activity (AA%), total phenols and flavonoids. Milk samples were inoculated, incubated, and the colonies were characterized as Staphylococcus aureus were adjusted to 1.0x106 UFC/mL in the 6 standard of the MacFarland scale. The sensitivity of the microbial isolates were determined in quintuplicate, in disk diffusion test. The minimum inhibitory concentration was determined by visible inhibition zone greater than 15mm. The results were evaluated by ANOVA, Tukey 5%, using the program SISVAR 5.3 - DEX/UFLA. Results implied that the aqueous extract from the bark of dried fruit was capable of inhibiting bacterial growth at concentrations of 3%. The other treatments only showed this activity, from the concentrations of 15%, 20% and 30% for dry EAF, in nature EAC and in nature EAF, respectively. Regarding the action antioxidant of dry EAC, was not correlated with total phenols and flavonoids. Probably other alkaloids substances present in the extract studied, may have been responsible for this activity. It is concluded that extracts of Punica granatum L., especially those obtained by the shell of the fruit, showed inhibitory activity against S. aureus, indicating your use potential for the control of bovine mastitis.

In vitro enamel remineralization by low-fluoride toothpaste with calcium citrate and sodium trimetaphosphate

The objective of this study was to evaluate in vitro the effect of a low fluoride toothpaste (450 μgF/g, NaF) combined with calcium citrate (Cacit) and sodium trimetaphosphate (TMP) on enamel remineralization. Bovine enamel blocks had the enamel surface polished sequentially to determine the surface hardness. After production of artificial carious lesions, the blocks selected by their surface hardness were submitted to remineralization pH cycling and daily treatment with dentifrice suspensions (diluted in deionized water or artificial saliva): placebo, 275, 450, 550 and 1,100 μgF/g and commercial dentifrice (positive control, 1,100 μgF/g). Finally, the surface and cross-section hardness was determined for calculating the change of surface hardness (%SH) and mineral content (%ΔZ). Fluoride in enamel was also determined. The data from %SH, %ΔZ and fluoride were subjected to two-way analysis of variance followed by Student-Newman-Keuls's test (p<0.05). The mineral gain (%SH and %ΔZ) was higher for toothpastes diluted in saliva (p<0.05), except for the 450 mgF/g dentifrice with Cacit/TMP (p>0.05). The 450 Cacit/ TMP toothpaste and the positive control showed similar results (p>0.05) when diluted in water. A dose-response was observed between fluoride concentration in toothpastes and fluoride present in enamel, regardless of dilution. It was concluded that it is possible to enhance the remineralization capacity of low F concentration toothpaste by of organic (Cacit) and inorganic (TMP) compounds with affinity to hydroxyapatite.

Efeito da interleucina-15 sobre a atividade fungicida, metabolismo oxidativo e produção de citocinas por monócitos humanos, infectados in vitro com Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Digestibilidade das proteínas do soro do leite mediante ensaio in vitro

Pós-graduação em Alimentos e Nutrição - FCFAR

Identificação e avaliação in vitro da atividade inibitória de Lactobacillus spp isolados do ingúvio e cecos de aves (Gallus gallus, Linneaus, 1758), em relação a sorotipos de Salmonella

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Otimização de protocolos de germinação in vitro de pinhão-manso (Jatropha curcas L.)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)