Antonio Olavo Cardoso Jorge

Fatec Sao Jose dos Campos, São José dos Campos, São Paulo, Brazil

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Publications (175)157.5 Total impact

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    ABSTRACT: The role of matrix metalloproteinases (MMPs) in tissue degradation has become evident in many diseases and great interest therefore exists in the pharmacological control of the activity of these enzymes. This study evaluated the effect of caffeic acid phenethyl ester (CAPE) on the production of MMPs and their inhibitor (TIMP) in monocytes activated by lipopolysaccharide (LPS). The human monocytic cell line (THP-1) was treated with non-cytotoxic concentrations of CAPE (10 and 60μM) combined with 1μg/mL of LPS. The gene expression of MMP-1, MMP-9 and TIMP-1 was evaluated by quantitative real-time polymerase chain reaction. The protein secretion into the culture medium was assessed via enzyme-linked immunosorbent assay and the gelatinolytic activity of MMP-9 by zymography. CAPE, especially at the highest concentration, down-regulated MMP-1 and MMP-9 gene expression but up-regulated the gene expression of TIMP-1. Furthermore, CAPE reduced the secreted protein level of MMP-1 and MMP-9 as well as the gelatinolytic activity of MMP-9. CAPE was able to inhibit the gene expression, production and the activity of MMPs induced by LPS and also increased the gene expression of TIMP-1. The present observations suggest that CAPE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is important for the control of different diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Archives of Oral Biology 05/2015; 60(9). DOI:10.1016/j.archoralbio.2015.04.009 · 1.88 Impact Factor
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    ABSTRACT: With the increasing number of strains of Candida ssp. resistant to antifungal agents, the accomplishment of researches that evaluate the effects of new therapeutic methods, like photodynamic inactivation (PDI), becomes important and necessary. Thus, the objective of this study was to verify the effects of the PDI on Candida albicans biofilms, evaluating their effects on the expression of the gene hydrolytic enzymes aspartyl proteinase (SAP5), lipase (LIP9), and phospholipase (PLB2). Clinical strains of C. albicans (n = 9) isolated from patient bearers of the virus HIV and a pattern strain ATCC 18804 were used. The quantification of gene expression was related to the production of hydrolytic enzymes using the quantitative polymerase chain reaction (qPCR) assay. For PDI, we used laser-aluminum-gallium arsenide low power (red visible, 660 nm) as a light source and the methylene blue at 300 μM as a photosensitizer. We assessed two experimental groups for each strain: (a) PDI: sensitization with methylene blue and laser irradiation and (b) control: without sensitization with methylene blue and light absence. The PDI decreased gene expression in 60 % of samples for gene SAP5 and 50 % of the samples decreased expression of LIP9 and PLB2. When we compared the expression profile for of each gene between the treated and control group, a decrease in all gene expression was observed, however no statistically significant difference (Tukey's test/p = 0.12). It could be concluded that PDI (photosensitization with methylene blue followed by low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.
    Lasers in Medical Science 04/2015; DOI:10.1007/s10103-015-1747-0 · 2.42 Impact Factor
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    ABSTRACT: Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.
    Brazilian Dental Journal 03/2015; 26(2):105-109. DOI:10.1590/0103-6440201300199
  • 03/2015; 119(3):e137. DOI:10.1016/j.oooo.2014.07.151
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    ABSTRACT: Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, few studies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. The objective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804 using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and on the capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluate the effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histological analysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillus culture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate were able to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C. albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus compared to the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans and protected G. mellonella against experimental candidiasis in vivo.
    Virulence 02/2015; 6(1). DOI:10.4161/21505594.2014.981486 · 3.32 Impact Factor
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    ABSTRACT: BACKGROUND: The search for alternative therapies for oral candidiasis is a necessity and the use of medicinal plants seems to be one of the promising solutions. The objective of this study was to evaluate the in vitro and in vivo effects of the essential oil of Melaleuca alternifolia on Candida albicans. METHODS: The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of M. alternifolia were determined by the broth microdilution assay. For the in vivo study, twelve immunosuppressed mice with buccal candidiasis received topical applications of M. alternifolia with MBEC. After treatment, yeasts were recovered from the mice and quantified (CFU/mL). Mice were killed for morphologic analysis of the tongue dorsum by optical and scanning electron microscopy. Data were analyzed using Student's t test or Mann-Whitney test. RESULTS: The MIC of M. alternifolia was 0.195% and the MBEC was 12.5%. Treatment with M. alternifolia achieved a 5.33 log reduction in C. albicans and reduced the microscopic lesions of candidiasis. CONCLUSIONS: M. alternifolia oil at a 12.5% was effective to eradicate a C. albicans biofilm formed in vitro and to reduce yeasts of C. albicans in an immunosuppressed mouse model.
    BMC Complementary and Alternative Medicine 12/2014; 14(1):489. DOI:10.1186/1472-6882-14-489 · 1.88 Impact Factor
  • 08/2014; 17(3). DOI:10.14295/bds.2014.v17i3.999
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    ABSTRACT: To evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7).
    Archives of Oral Biology 05/2014; 59(8):808-814. DOI:10.1016/j.archoralbio.2014.05.013 · 1.88 Impact Factor
  • 05/2014; 117(5):e383-e384. DOI:10.1016/j.oooo.2014.01.197
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    ABSTRACT: Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 μM) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.
    Lasers in Medical Science 04/2014; DOI:10.1007/s10103-014-1570-z · 2.42 Impact Factor
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    ABSTRACT: Adhesion and colonization of the oral cavity by Candida albicans is an initial step in candidosis. Orthodontic and other oral appliances seem to favor candidal presence. The aim of this work was to compare the presence of Candida species in saliva, their adherence to oral epithelial cells, and the levels of anti-C. albicans IgA in children with or without orthodontic appliances. This study included 30 children 5 to 12 years old (9.1 ± 1.7 years old) who were users of removable orthodontic devices for at least 6 months and 30 control children of similar ages (7.7 ± 1.5 years old). The presence of yeast species in the saliva was evaluated by microbiological methods. Candida species were identified using phenotypic methods. Anti-C. albicans IgA levels in saliva were analyzed by ELISA. The yeasts adhering to oral epithelial cells were assessed by exfoliative cytology. No statistically significant differences were observed for saliva yeast counts and anti-C. albicans IgA levels between the studied groups. Children with orthodontic devices exhibited more yeast cells adhering to oral epithelial cells and a higher percentage of non-albicans species relative to the control group. In conclusion, orthodontic appliances may favor the adherence of Candida to epithelial cells but do not influence the presence of these yeasts in saliva, and the levels of anti-C. albicans IgA do not correlate with yeast adherence or presence of Candida in the oral cavity.
    Brazilian oral research 12/2013; 28(1). DOI:10.1590/S1806-83242013005000031 · 0.77 Impact Factor
  • 10/2013; 16(3). DOI:10.14295/bds.2013.v16i3.909
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    ABSTRACT: The aim of this study was to evaluate the effects of Citrus limonum and Citrus aurantium essential oils (EOs) compared to 0.2% chlorhexidine (CHX) and 1% sodium hypochlorite (NaOCl) on multi-species biofilms formed by Candida albicans, Enterococcus faecalis and Escherichia coli. The biofilms were grown in acrylic disks immersed in broth, inoculated with microbial suspension (106 cells/mL) and incubated at 37°C / 48 h. After the biofilms were formed, they were exposed for 5 minutes to the solutions (n = 10): C. aurantium EO, C. limonum EO, 0.2% CHX, 1% NaOCl or sterile saline solution [0.9% sodium chloride (NaCl)]. Next, the discs were placed in sterile 0.9% NaCl and sonicated to disperse the biofilms. Tenfold serial dilutions were performed and the aliquots were seeded onto selective agar and incubated at 37°C / 48 h. Next, the number of colony-forming units per milliliter was counted and analyzed statistically (Tukey test, p ≤ 0.05). C. aurantium EO and NaOCl inhibited the growth of all microorganisms in multi-species biofilms. C. limonum EO promoted a 100% reduction of C. albicans and E. coli, and 49.3% of E. faecalis. CHX was less effective against C. albicans and E. coli, yielding a reduction of 68.8% and 86.7%, respectively. However, the reduction of E. faecalis using CHX (81.7%) was greater than that obtained using C. limonum EO. Both Citrus limonum and Citrus aurantium EOs are effective in controlling multi-species biofilms; the microbial reductions achieved by EOs were not only similar to those of NaOCl, but even higher than those achieved by CHX, in some cases.
    Brazilian oral research 10/2013; DOI:10.1590/S1806-83242013005000024 · 0.77 Impact Factor
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    ABSTRACT: An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.
    Brazilian Journal of Oral Sciences 09/2013; 12(3):199-204. DOI:10.1590/S1677-32252013000300009
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    ABSTRACT: Candida albicans is an opportunistic yeast that can cause oral candidosis through the formation of a biofilm, an important virulence factor that compromises the action of antifungal agents. The objective of this study was to compare the effect of rose bengal (RB)- and eosin Y (EY)-mediated photodynamic inactivation (PDI) using a green light-emitting diode (LED; 532 ± 10 nm) on planktonic cells and biofilms of C. albicans (ATCC 18804). Planktonic cultures were treated with photosensitizers at concentrations ranging from 0.78 to 400 μM, and biofilms were treated with 200 μM of photosensitizers. The number of colony-forming unit per milliliter (CFU/mL) was compared by analysis of variance and Tukey's test (P ≤ 0.05). After treatment, one biofilm specimen of the control and PDI groups were examined by scanning electron microscopy. The photosensitizers (6.25, 25, 50, 200, and 400 μM of EY, and 6.25 μM of RB or higher) significantly reduced the number of CFU/mL in the PDI groups when compared to the control group. With respect to biofilm formation, RB- and EY-mediated PDI promoted reductions of 0.22 log10 and 0.45 log10, respectively. Scanning electron microscopy showed that the two photosensitizers reduced fungal structures. In conclusion, EY- and RB-mediated PDI using LED irradiation significantly reduced C. albicans planktonic cells and biofilms.
    Lasers in Medical Science 09/2013; 29(3). DOI:10.1007/s10103-013-1435-x · 2.42 Impact Factor
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    ABSTRACT: Objective: Evaluating the ability of different auxiliary chemicals (1% sodium hypochlorite, 2% chlorhexidine gel and 12% propolis extract) to neutralize cytotoxic effects of LTA of Enterococcus faecalis in root canals, analyzing the production of nitric oxide and cytokines (IL-1β, IL-6, TNF-α) by macrophages. Method: Forty single-rooted roots (16 mm) were used. The canals were prepared to working length (15 mm) and distributed in 4 microplates. After sterilization (Co60 gamma radiation), the root canals were inoculated with 10 uL of E. faecalis LTA (repeated 3 times every 24 hours). The instrumentation was performed with five titanium nickel rotary instruments and, according to the irrigant, the specimens were divided into four groups (n=10): NaOCl) 1% sodium hypochlorite; CLX) 2% chlorhexidine gel; PRO) 12% glycolic extract of propolis; CONTROL) physiologic saline. The collect was performed immediately after instrumentation. Macrophages (RAW 264.7) were activated with collected samples and, after 24 hours, the supernatants were used to verify the production of nitric oxide (Griess reagent) and cytokines (IL-1β, IL-6, TNF-α) by enzyme immunoassay (ELISA). The results were analyzed by ANOVA, Tukey test, 5%. Result: NaOCl, PRO and CLX groups showed statistically lower values of nitric oxide in relation to the CONTROL group (p<0.05). NaOCl and PRO groups had lower values of IL-1β and IL-6, being similar with each other (p> 0.05) and different from CLX and CONTROL (p<0.05). All groups showed similar results of TNF-α production (p>0.05). Conclusion: Sodium hypochlorite, chlorhexidine gel and propolis extract were able to neutralize the different cytotoxic effects of LTA in root canals, however, propolis extract and sodium hypochlorite were the most effective.
    Annual Meeting of the IADR Continental European Division 2013; 09/2013
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    ABSTRACT: Interactions between fungi and bacteria are present in nature and have great medical and environmental importance. Studies aimed to characterize these interactions are essential for understanding the pathogenesis of diseases and discovery of new therapeutic strategies. Objective: To evaluate the microbial interaction between S. mutans and C. albicans by means of in vitro study. Method: It was analyzed the effects of S. mutans in biofilm formation and filamentation by C. albicans. In both tests, was evaluated the direct effects of the cells of S. mutans and also its indirect effects, using the supernatant of this culture. Monotypic biofilms of C. albicans (ATCC 18804) and heterotypic of C. albicans and S. mutans (UA 159) were formed at the bottom of microtiter plate 96-wells and was quantified by calculating CFU / mL. The filamentation was visualized in optical microscopy after incubation for 24 h in microaerophilia. For results analysis, were used ANOVA and Tukey's test for CFU / mL data of the biofilm formation and the Kruskal-Wallis test for filamentation. Result: Regarding to the study of biofilm, it was found that cells of S.mutans favored biofilm formation by C. albicans, however, when C. albicans was placed in contact only with the supernatant of the culture of S. mutans, was decreased 0.27 log10 of biofilm formed by C. albicans in comparison with the control group (P= 0,0002). In the study of inhibition was also observed filamentation of morphological transition of C. albicans in the presence of supernatant of S. mutans, which was statistically significant compared with the control group (P= 0,0003). Conclusion: Based on these results, it was concluded that S. mutans secretes byproducts into the culture medium in vitro, showing inhibitory effects on C. albicans, interfering in their ability to filamentation and biofilm formation.
    Annual Meeting of the IADR Continental European Division 2013; 09/2013
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    ABSTRACT: Objective: C. albicans is the predominant species associated with mucosal and systemic fungal infections. Nevertheless, the epidemiology of yeast infection is rapidly evolving and non-albicans Candida species have emerged as important pathogens, occurring both singly or in mixed species infections, often with C. albicans. Then, this study evaluated the interactions of C. albicans with C. glabrata and C. krusei in the experimental mixed candidiasis using invertebrate and vertebrate models hosts. Method: Single or mixed experimental candidiasis were induced in Galleria mellonella larvae and immunosuppressed mice by inoculation of homotypic or heterotypic microbial suspensions of the strains C. albicans ATCC 18804, C. glabrata ATCC 90030 and C. krusei ATCC 6258. Experimental candidiasis in the invertebrate model of G. mellonella was evaluated by the survival rate using Log-rank test. On the other hand, experimental candidiasis in the immunosuppressed mice model was studied in the oral cavity by counting of CFU/mL and the data were analysed by ANOVA and Tukey test. Result: In the G. mellonella model, 100% of the larvae died within 18 h of single infection with C. albicans. However, when G. mellonella larvae were infected by heterotypic microbial suspension, 100% of mortality occurred 96 and 72 h after the mixed infection by C. albicans - C. krusei (p=0.0001) and C. albicans - C. glabrata (p=0.0001) respectively. The number of C. albicans (CFU/mL) recovered from the oral cavity of the immunosuppressed mice was higher for the single infection (5.75 log10) compared to mixed infections with C. glabrata (5.46 log10, p=0.014) or C. krusei (5.32 log10,p=0.014). Conclusion: In both hosts models studied, single infection by C. albicans was more intense than mixed infections of C. albicans with non-albicans Candida species, suggesting that C. albicans established competitive interactions with C. glabrata and C. krusei during the development of experimental candidiasis.
    Annual Meeting of the IADR Continental European Division 2013; 09/2013
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    ABSTRACT: Objective: To evaluate in vivo the antimicrobial action of 12% propolis glycolic extract compared with 1% sodium hypochlorite and 2% chlorhexidine gel used as irrigant during treatment of root canals with primary infection and periapical lesions. The identification of the microorganisms related to the infections was also investigated. Method: Thirty teeth with pulp necrosis and periapical lesions were selected. The root canals were instrumented and divided into 3 groups (n=10), according to the irrigant used during instrumentation: NaOCl- 1% sodium hypochlorite; CLX- 2% chlorhexidine gel; and PRO- 12% propolis glycolic extract. Three samples were taken from root canals: S1) immediately after coronary opening; S2) after instrumentation; S3) after 14 days of dressing (calcium hydroxide + propylene glycol). The samples were seeded in different culture media, under aerobic and anaerobic conditions. DNA was extracted from canal samples and subjected to polymerase chain reaction (PCR). The results were submitted to statistical tests of Kruskal Wallis and Dunn (5%). Result: Microbial growth was observed in 100% of the S1. There was a decrease in quantity of microorganisms in S2 and S3 in relation to S1 (p<0.05) for all irrigation substances evaluated. Among the microorganisms identified, the most common species were Parvimonas micra, Tannerella forsythia, Porphyromonas endodontalis and Prevotella nigrescens. Conclusion: The 12% propolis glycolic extract might be indicated as irrigant in the treatment of teeth with primary endodontic infections and periapical lesions, since it showed similar antimicrobial activity of 1% sodium hypochlorite and 2% chlorhexidine gel. The most prevalent species were Parvimonas micra, Tannerella forsythia, Porphyromonas endodontalis and Prevotella nigrescens.
    Annual Meeting of the IADR Continental European Division 2013; 09/2013
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    ABSTRACT: Objective: In the oral cavity, C. albicans and non-albicans species reside in a complex and dynamic structure called biofilm. The objective of this study was investigate the expression of genes TEC-1 and EFG-1 of C. albicans at 24 and 48 hours in mixed biofilms with C. krusei by PCR in Real Time. Method: Standardized suspension of C. albicans (ATCC 18804) at a concentration of 107 cells/mL was added to wells of 24-well plates, and incubated under shaking at 37°C for 90 min. Subsequently was added C. krusei (ATCC 6258) at the same concentration. The plates containing the two micro-organisms were incubated under shaking at 37°C for 48h. After the passed time, removed the entire contents of the well and added to 1 mL of Trizol RNA extraction. This was measured and transcribed by Kit SuperScript ™ III First-Strand Synthesis Supermix for qRT-PCR (Invitrogen) and cDNA obtained was frozen at -20 ˚ C. For Real-Time amplification used the kit SYBR® Green qPCR Supermix-UDG (Invitrogen) with primers for genes TEC-1 and EFG-1 were normalized by the housekeeping Actin-1 gene. Result: The target genes, TEC-1 and EFG-1obtained a significant reduction in gene expression in different time in heterotypic biofilms in relation to monotypic (p <0.05). Conclusion: The reduction obtained in gene expression in heterotypic biofilms suggests an antagonistic/competitive relationship of C.krusei when associated with C. albicans.
    Annual Meeting of the IADR Continental European Division 2013; 09/2013

Publication Stats

1k Citations
157.50 Total Impact Points

Institutions

  • 2001–2015
    • Fatec Sao Jose dos Campos
      São José dos Campos, São Paulo, Brazil
  • 2000–2015
    • São Paulo State University
      • • Department of Microbiology and Immunology
      • • Department of Dental Materials and Prosthodontics
      San Paulo, São Paulo, Brazil
  • 2010–2012
    • CEP America
      Emeryville, California, United States
  • 2007–2011
    • University of São Paulo
      San Paulo, São Paulo, Brazil
  • 2004–2011
    • University of Campinas
      Conceição de Campinas, São Paulo, Brazil
  • 2002–2010
    • Universidade de Taubaté
      • Departamento de Odontologia
      Taubaté, São Paulo, Brazil
  • 2008–2009
    • Universidade Federal de Santa Maria
      • Department of Stomatology
      Santa Maria, Estado do Rio Grande do Sul, Brazil
  • 2003
    • Università degli Studi di Torino
      Torino, Piedmont, Italy