Síntese, caracterização e atividade antimicrobiana de nanofibras de policaprolactona - nistatina produzidas por eletrofiação / Synthesis, characterization and antimicrobial properties of nanofibers polycaprolactane - nystatin produced by electrospinning

O objetivo desse estudo foi sintetizar um scaffold de nanofibras (NFs) de Policaprolactona (PCL) com adição de nistatina (NYS), caracterizar e avaliar a atividade antimicrobiana. NFs foram sintetizadas pela técnica da eletrofiação, utilizando-se solução de PCL puro e PCL adicionado de NYS. Para a síntese das NFs de PCL, o polímero foi dissolvido em Dimetilformamida (DMF) e 1,1,2,2 Tetracloroetano (TCE), logo após foram adicionadas três concentrações de NYS (Grupos A, B e C). As amostras foram analisadas em Microscopia Eletrônica de Varredura (MEV), Espectroscopia por energia dispersiva (EDS), Análise de molhabilidade, Análise de Difratometria de Raios-X (DRX) e Espectroscopia de Infravermelho por Transformada de Fourier (FTIR). Após a análise em MEV foi realizada a média dos diâmetros das fibras com Software ImageJ. As atividades antimicrobianas foram avaliadas por meio dos testes de concentração inibitória mínima (CIM) e concentração microbicida mímica (CMM). As NFs de PCL com NYS (A) apresentaram diâmetro de 0,99µm ±0,29, o padrão do grupo B apresentou uma média de diâmetro de 1,3µm ±0,56 e o grupo C 1,24µm ±0,48. O DRX e o EDS comprovaram a presença de PCL e de NYS nas amostras. A CIM e a CMM comprovaram a ação fungicida e fungistática das NFs A, B e C(AU)

The aim of this study was to synthesize a scaffold of policaprolactane (PCL) nanofibers (NFs) with addition of nystatin (NYS), to characterize and evaluate the antimicrobial activity. NFs were synthesized by the electro-spinning technique, using pure PCL and NYS and PCL solutions. For the synthesis of PCL NFs, the polymer was dissolved in Dimethylformamide (DMF) and 1,1,2,2- Tetrachloroethane (TCE), after which three concentrations of NYS (Groups A, B and C) were added. The samples were analyzed in Scanning Electron Microscopy (SEM), Dispersive Energy Spectroscopy (EDS), Goniometer, X-ray Diffraction Analysis (XRD) and Fourier Transform Infrared Spectroscopy (FTIR). After SEM analysis, the mean fiber diameter was performed with ImageJ Software. The antimicrobial activities were evaluated through the tests of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC).The NFs of PCL with NYS (A) had a diameter of 0.99 µm ± 0.29, the standard of group B presented a mean diameter of 1.3 µm ± 0.56 and group C 1.24 µm ± 0.48. The DRX and the EDS verified the presence of PCL and NYS in the samples. CIM and CMM proved the fungicidal and fungistatic action of NFs A, B and C(AU)

Propofol, fentanyl and remifentanil do not impair the phagocytic capacity to of human peripheral blood polimorphonuclear cells / Propofol, fentanil y remifentanil no alteran la capacidad de las células polimorfonucleares humanas de fagocitar a la Candida albicans

OBJECTIVES: The capacity of polymorphonuclear cells (PMN) to phagocyte microorganisms is an important function to be preserved during surgical interventions. Therefore, the aim of this study was to determine the effect of propofol, fentanyl and remifentanil combination on Candida albicans engulfment by human PMN. MATERIALS AND METHODS: Twenty patients scheduled to undergo surgical interventions (ASA I-II) received propofol, fentanyl and remifentanil as intravenous anesthesia. PMNs were obtained before and after the surgical procedure and phagocytosis assay was performed using opsonized C. albicans. RESULTS AND CONCLUSIONS: No differences between the values obtained before and after anesthesia treatment in the number of phagocytic PMN and the number of C. albicans engulfed were observed. These results suggest that the used anesthesic protocol does not alter one of the most important immune mechanisms.

OBJETIVOS: La capacidad de las células polimorfonucleares (CPN) de fagocitar a los microorganismos es una importante función que se debe de preservar durante las intervenciones quirúrgicas. Por lo tanto, el propósito de este estudio fue determinar el efecto de la combinación del propofol, el fentanil y el remifentanil en la ingestión de Candida albicans por parte de las CPN humanas. MATERIALES Y MÉTODOS: Veinte pacientes sujetos a intervenciones quirúrgicas (ASA I-II) recibieron propofol, fentanil y remifentanil como anestesia endovenosa. Los CPN se obtuvieron antes y después del procedimiento quirúrgico y el ensayo de fagocitosis fue realizando usando C. albicans opsonizada. RESULTADOS Y CONCLUSIONES: No se observaron diferencias significativas en los valores obtenidos antes y después del tratamiento anestésico tanto en el número de CPN fagocíticas como en el número de C. albicans dentro de las células. Estos hallazgos sugieren que el protocolo anestésico usado no altera uno de los mecanismos de defensa más importante del organismo.

Desenvolvimento de formulações probióticas para a prevenção da candidose bucal: estudo in vitro e in vivo / Development of probiotic formulations for prevention of oral candidiasis: in vitro and in vivo study

Os probióticos são considerados uma alternativa potencial para o controle da candidose, no entanto, existe uma falta de produtos probióticos direcionados para a cavidade bucal. Neste estudo, desenvolvemos formulações probióticas usando gellan gum, um biopolímero natural usado como aditivo alimentar, e investigamos os efeitos dessas formulações em Candida albicans. Para isso, Lactobacillus paracasei 28.4, uma cepa recentemente isolada da cavidade bucal, foi incorporada em várias concentrações de gellan gum (1 a 0,6%). Todas as concentrações testadas foram capazes de incorporar as células de L. paracasei, mantendo a viabilidade bacteriana. As formulações probióticas permaneceram estáveis por 7 dias quando armazenadas à temperatura ambiente ou a 4°C. Entretanto, o armazenamento a longo prazo das formulações probióticas foi conseguido apenas quando L. paracasei 28.4 foi liofilizado. As formulações probióticas proporcionaram uma liberação de células de L. paracasei por 24 horas, o que foi suficiente para inibir o crescimento de C. albicans com efeitos dependentes das concentrações celulares incorporadas no gellan gum. As formulações probióticas também tiveram atividade inibitória contra os biofilmes de Candida, reduzindo o número de células de Candida (p<0,0001), diminuindo a biomassa total (p=0,0003) e prejudicando a formação de hifas (p=0,0002) em relação ao grupo controle não tratado. Contudo, a formulação probiótica de gellan gum a 1% proporcionou uma colonização oral de L. paracasei em camundongos com aproximadamente 6 log de UFC/mL após 10 dias. Essa formulação inibiu o crescimento de C. albicans (p<0,0001), impediu o desenvolvimento de lesões de candidose (p=0,0013) e suprimiu a inflamação (p = 0,0006) quando comparada aos camundongos não tratados. Estes resultados indicam que o gellan gum pode ser um biomaterial promissor como sistema transportador de probióticos para prevenir a candidose oral(AU)

Probiotics are considered a potential alternative for the control of candidiasis, however there is a lack of probiotic products targeted for the oral cavity. In this study, we developed probiotic formulations using gellan gum, a natural biopolymer used as a food-additive, and investigated the effects of this delivery method on Candida albicans. Lactobacillus paracasei 28.4, a strain recently isolated from the oral cavity, was incorporated in several concentrations of gellan gum (1 to 0.6%). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic-gellan formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterial impregnated gellan gum could be achieved when L. paracasei 28.4 was lyophilized. The probiotic-gellan formulations provided a release of L. paracasei cells over 24 hours that was sufficient to inhibit the growth of C. albicans with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan formulations also had inhibitory activity against Candida biofilms by reducing the number of Candida cells (p < 0.0001), decreasing the total biomass (p = 0.0003) and impairing hyphae formation (p = 0.0002) in relation to the control group, not treated. However, only probiotic formulation of gellan gum 1% provided an oral colonization of L. paracasei in mice with approximately 6 log of CFU/mL after 10 days. This formulation inhibited the C. albicans growth (p < 0.0001), prevented the development of candidiasis lesions (p = 0.0013), and suppressed the inflammation (p = 0.0006) when compared to the mice not treated. These results indicate that gellan gum is a promising biomaterial as a carrier system of probiotics to prevent oral candidiasis(AU)

Influence of Acrylic Resin Denture Base Soaking Length in Siwak Extract Solution (Salvadora persica) on the Growth of Candida albicans

Objective: To know the influence of immersion length of denture base of acrylic resin in Siwak solution (Salvadora persica) on Candida albicans growth. Material and Methods: This type of research is laboratory experimental. The Siwak plant (Salvadora persica) was extracted and 1% solution was formed. The media used were Sabouraud Dextrose Broth (SDB) and Sabouraud Dextrose Agar (SDA). Candida albicans was cultured as 1 dose in 100 ml of SDB medium, and then incubated at shaker rotation for 1x24 hours. The concentration chosen to test the effectiveness of siwak extract solution was 1%, 10%, 15% and 25%. Data were presented as mean ± SD. Results: Zone of 6 hours Siwak extract immersion inhibitor by 43.47 ± 0.35, 8 hours to know the influence of immersion length of denture base of acrylic resin in Siwak solution (Salvadora persica) on candida albicans growth 44.42 ± 0.02, 10 hours of 52.79 ± 0.03. Conclusion: There is a difference of immersion length of denture base of acrylic resin on Candida albicans growth in Siwak extract solution (Salvadora persica).

Antifungal Activity, Phytochemical Characterization and Thermal Profile of Anadenanthera colubrina (Vell. ) Brenan

Objective: To investigate the antifungal potential of A. colubrina, and its phytochemical characteristics, thermal profile and toxicity. Material and Methods: To assess potential antifungal activity, the technique of microdilution was used with the determination of the Minimum Inhibitory Concentration and Minimum Fungicidal Concentration, using standard species of Candida and recent clinical isolates of Candida albicans. Analyses of action of the extract were performed on the wall and cell morphology of C. albicans, of the interactive effect between the plant extract and nystatin on C. albicans through the checkerboard method, and of growth kinetics. The phytochemical screening was determined by spectrophotometry. The thermal profile was traced with the determination of thermogravimetric curves (TG) and differential scanning calorimetry (DSC). The toxicity was evaluated by the method of hemolysis. Results: The extract of A. colubrina showed a fungistatic potential against all bacteria tested and it acted by modifying the cellular morphology of C. albicans. There was a synergism between nystatin and the plant extract (FIC=0.375), and 53.18% of total polyphenols were determined. The TG curve showed the occurrence of three steps of thermal decomposition. None of the tested concentrations became the effective cytotoxic concentration. Conclusion: Further studies should be conducted to understand the efficacy and the mechanisms of action involved in the antifungal activity of the plant extract of A. colubrina in order to produce a new drug for the treatment of oral candidiasis.

Expressão dos genes ALS3, HWP1, BCR1, TEC1, CPH1 e EFG1 de Candida albicans em biofilmes após inativação fotodinâmica / Expression of the Candida albicans genes ALS3, HWP1, BCR1, TEC1, CPH1 and EFG1 in biofilms after photodynamic inactivation

Os micro-organismos estão se tornando cada vez mais resistentes aos antimicrobianos e cepas de Candida albicans resistentes aos antifúngicos tem sido isoladas, assim, torna-se importante e necessário a realização de pesquisas que avaliem os efeitos de novos métodos terapêuticos, como a inativação fotodinâmica antimicrobiana (aPDI). Assim, o objetivo deste estudo foi verificar os efeitos da inativação fotodinâmica sobre biofilmes de Candida albicans, avaliando seus efeitos sobre a expressão dos genes TEC1 (fator de transcrição), HWP1 (proteína de parede celular das hifas), EFG1 (regulador transcricional relacionado com a morfogênese), BCR1 (regulador da formação de biofilme e da parede celular), CPH1 (regulador transcricional envolvido na morfogênese) e ALS3 (adesina) de C. albicans. Foram avaliadas 30 amostras isoladas de pacientes portadores de HIV e 30 amostras de pacientes com estomatite protética, quanto a produção de biofilme, peso seco e filamentação. Destas, foram selecionadas as amostras mais virulentas de cada grupo que apresentaram melhor capacidade de formação de biofilme e filamentação. Assim, foi utilizada uma amostra clínica de C. albicans isolada de paciente portador de HIV, uma amostra clínica de C. albicans isolada de paciente com estomatite protética e uma cepa padrão ATCC 18804. A quantificação da expressão dos genes foi relacionada à produção desses genes nas amostras clínicas e na cepa de referência utilizando-se ensaio de PCR em tempo real. Para a aPDI, foram utilizados os fotossensibilizadores azul de metileno a 300 µM e eritrosina a 400 µM sensibilizados com laser de Índio-Gálio-Alumínio-Fósforo de baixa potência (vermelho visível, 660 nm) e LED verde (532 ± 10 nm), respectivamente. Foram avaliados quatro grupos experimentais para a aPDI: a) F+L+: sensibilização com o corante e irradiação com luz; b) F+L-: somente tratamento com o fotossensibilizador; c) F-L+: somente irradiação com luz e d) F-L-: sem sensibilização com o corante e ausência de luz. Os resultados foram analisados por t-test, com um nível de significância de 5%. Após a análise fenotípica, as amostras Ca30 e 39 S foram selecionadas para a realização da aPDI. Como esperado, apenas para o grupo F+L+, quando comparado com o grupo F-L-, todos os genes analisados foram sub expressos após a aPDI. O fold-decrease para os genes ALS3, HWP1, BCR1, TEC1, CPH1 e EFG1 foram 0,73; 0,39; 0,77; 0,71; 0,67 e 0,60; para laser, respectivamente, e 0,66; 0,61; 0,50; 0,43; 0,54 e 0,66; para LED, respectivamente. Pode-se concluir que a aPDI mostrou uma redução na expressão dos genes de C. albicans, sugerindo a diminuição de sua virulência(AU)

Os micro-organismos estão se tornando cada vez mais resistentes aos antimicrobianos e cepas de Candida albicans resistentes aos antifúngicos tem sido isoladas, assim, torna-se importante e necessário a realização de pesquisas que avaliem os efeitos de novos métodos terapêuticos, como a inativação fotodinâmica antimicrobiana (aPDI). Assim, o objetivo deste estudo foi verificar os efeitos da inativação fotodinâmica sobre biofilmes de Candida albicans, avaliando seus efeitos sobre a expressão dos genes TEC1 (fator de transcrição), HWP1 (proteína de parede celular das hifas), EFG1 (regulador transcricional relacionado com a morfogênese), BCR1 (regulador da formação de biofilme e da parede celular), CPH1 (regulador transcricional envolvido na morfogênese) e ALS3 (adesina) de C. albicans. Foram avaliadas 30 amostras isoladas de pacientes portadores de HIV e 30 amostras de pacientes com estomatite protética, quanto a produção de biofilme, peso seco e filamentação. Destas, foram selecionadas as amostras mais virulentas de cada grupo que apresentaram melhor capacidade de formação de biofilme e filamentação. Assim, foi utilizada uma amostra clínica de C. albicans isolada de paciente portador de HIV, uma amostra clínica de C. albicans isolada de paciente com estomatite protética e uma cepa padrão ATCC 18804. A quantificação da expressão dos genes foi relacionada à produção desses genes nas amostras clínicas e na cepa de referência utilizando-se ensaio de PCR em tempo real. Para a aPDI, foram utilizados os fotossensibilizadores azul de metileno a 300 µM e eritrosina a 400 µM sensibilizados com laser de Índio-Gálio-Alumínio-Fósforo de baixa potência (vermelho visível, 660 nm) e LED verde (532 ± 10 nm), respectivamente. Foram avaliados quatro grupos experimentais para a aPDI: a) F+L+: sensibilização com o corante e irradiação com luz; b) F+L-: somente tratamento com o fotossensibilizador; c) F-L+: somente irradiação com luz e d) F-L-: sem sensibilização com o corante e ausência de luz. Os resultados foram analisados por t-test, com um nível de significância de 5%. Após a análise fenotípica, as amostras Ca30 e 39 S foram selecionadas para a realização da aPDI. Como esperado, apenas para o grupo F+L+, quando comparado com o grupo F-L-, todos os genes analisados foram sub expressos após a aPDI. O fold-decrease para os genes ALS3, HWP1, BCR1, TEC1, CPH1 e EFG1 foram 0,73; 0,39; 0,77; 0,71; 0,67 e 0,60; para laser, respectivamente, e 0,66; 0,61; 0,50; 0,43; 0,54 e 0,66; para LED, respectivamente. Pode-se concluir que a aPDI mostrou uma redução na expressão dos genes de C. albicans, sugerindo a diminuição de sua virulência(AU)

Isolamento e caraterização de Lactobacillus spp. da cavidade bucal e sua ação probiótica sob Candida albicans: formação de biofilme, infecção em modelos de invertebrados e expressão dos genes EFG1, HWP1 e ALS1 / Isolation and characterization of Lactobacillus spp. from oral cavity and their probiotic action in Candida albicans: biofilm formation, infection in invertebrate models and EFG1, HWP1 and ALS1 gene expression

O estudo da atividade inibitória de Lactobacillus pode contribuir na descoberta de novas estratégias terapêuticas nas infecções por Candida. Nesse contexto, o objetivo desse estudo foi isolar e identificar Lactobacillus da cavidade bucal de indivíduos livres de cárie e avaliar seu potencial de inibição de C. albicans por meio de estudos in vitro e in vivo. Primeiramente, foram avaliados os efeitos de 30 isolados clínicos de Lactobacillus sobre o número de células viáveis (UFC) em biofilme de C. albicans e sobre a formação de hifas. Os isolados que obtiveram os maiores efeitos inibitórios sobre C. albicans foram selecionados para os testes de determinação da biomassa total dos biofilmes pela absorbância do cristal violeta, análise da arquitetura dos biofilmes por microscopia eletrônica de varredura (MEV) e quantificação da expressão de genes de C. albicans (ALS3, HWP1, EFG1 e CPH1) por qPCR. Esses isolados também foram submetidos a estudos in vivo usando os modelos de Galleria mellonella e Caenorhabditis elegans. Para o estudo em G. mellonella, a infecção experimental foi avaliada pela curva de sobrevivência, quantificação da carga fúngica na hemolinfa, densidade hemocitária, quantificação da expressão gênica de peptídeos antifúngicos (Gallerymicina e Galiomicina) e monitoramento da infecção de C. albicans por análise de bioluminescência. No modelo de C. elegans, a infecção foi avaliada por meio dos ensaios de curva de sobrevivência e estudo da filamentação de C. albicans. Os resultados dos ensaios in vitro demonstraram que L. paracasei 28.4, L. rhamnosus 5.2 e L. fermentum 20.4 foram as cepas com maior atividade antimicrobiana sobre os biofilmes de C. albicans. Nessas cepas, todos os genes analisados foram regulados negativamente na associação com Lactobacillus quando comparados com o grupo controle. No estudo in vivo, a injeção de L. paracasei 28.4 em G. mellonella infectadas com C. albicans aumentou a sobrevida das larvas, o número de hemócitos e a expressão de peptídeos antifúngicos, reduzindo assim a UFC de C. albicans. Em C. elegans, L. paracasei 28.4 também foi capaz de aumentar a sobrevida dos vermes infectados com C. albicans e reduzir a filamentação. Conclui-se que L. fermentum 20.4, L. paracasei 28.4 e L. rhamnosus 5.2 tem potencial para serem usados como probióticos na cavidade bucal devido sua ação anti-biofilme e sua regulação negativa dos genes de virulência de C. albicans. L. paracasei 28.4 foi capaz de prolongar a sobrevida nos dois modelos experimentais infectados com C. albicans por apresentarem ação antifúngica e imunomodulatória(AU)

The study of the antifungal activity of Lactobacillus may contribute to the discovery of new therapeutic strategies for Candida infections. In this context, the objective of this study was to isolate and identify Lactobacillus from the oral cavity of caries-free subjects and to evaluate its effects through in vitro and in vivo studies. First, the effects of 30 clinical isolates of Lactobacillus on the number of viable cells (CFU) in biofilms of C. albicans and on hyphae formation were evaluated. The isolates that obtained the highest inhibitory effects on C. albicans were selected for biofilm biomass determination by violet crystal absorbance, analysis of biofilm architecture by scanning electron microscopy (SEM) and quantification of the expression of C. albicans (ALS3, HWP1, EFG1 and CPH1) by real time PCR. These isolates were also submitted to in vivo studies using the Galleria mellonella and Caenorhabditis elegans models. For the study in the model of Galleria mellonella, the experimental infection was evaluated by the survival curve, quantification of the fungal load in the hemolymph, hemocitary density, the gene expression of antifungal peptides (Gallerymicin and Galiomicin) and monitoring of C. albicans infection by bioluminescence analysis. In the Caenorhabditis elegans model, the infection was evaluated by the survival curve assays and the study of C. albicans filamentation. The results of in vitro tests demonstrated that L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 were the strains with the highest antimicrobial activity on the biofilms of C. albicans. In these strains, all analyzed genes were negatively regulated in association with Lactobacillus when compared to the control group. In the in vivo study, the injection of L. paracasei 28.4 into the G. mellonella increased survival of the larvae, the number of hemocytes and the expression of antifungal peptides, thus reducing the CFU of C. albicans. In C. elegans, L. paracasei 28.4 was also able to increase the survival of worms infected with C. albicans and reduce the filamentation. We conclude that L. fermentum 20.4, L. paracasei 28.4 and L. rhamnosus 5.2 have potential to be used as probiotics in the oral cavity due to their anti-biofilm action and their negative regulation of virulence genes of C. albicans. L. paracasei 28.4 was able to prolong survival of both experimental models infected with C. albicans for having antifungal and immunomodulatory action(AU)

Potencial antimicrobiano dos antissépticos de uso popular anapyon®, água rabelo® e malvatricin® sobre microrganismos do meio ambiente oral / Antimicrobial Potential of the Popular Antiseptics Anapyon®, Água Rabelo® and Malvatricin® against Oral Microorganisms

Objetivo: Verificar, ?in vitro?, o potencial antimicrobiano do Anapyon®, da Água Rabelo® e do Malvatricin® sobre microrganismos presentes na cavidade oral.Método: Para realização do experimento, utilizou-se o protocolo sequenciado durante quatro dias que avaliou, através da medida da densidade óptica, o potencial antimicrobiano dos fármacos nos microrganismos (Staphylococcus aureus, Candida tropicalis, Candida parapsilosis e Candida albicans), em suas formas planctônicas, apenas o Malvatricin® foi avaliado sobre as formas de biofilme por ser o único fármaco que apresentou resultados satisfatórios sobre as formas planctônicas. O estudo adotou como controle negativo a água destilada e controle positivo a Clorexidina®. Os resultados obtidos foram submetidos a uma análise estatística com os testes de Kruskal-Wallis e Mann-Whitney. Resultados: Observou-se, através da medida da densidade óptica, que apenas o Malvatricin® apresentou bons resultados na forma planctônica, sendo estes semelhantes ao controle positivo (clorexidina), considerado padrão nos ensaios antimicrobianos em Odontologia. Os resultados do Malvatricin® foram estatisticamente melhores quando comparados aos demais fármacos (Anapyon®, Água Rabelo®) e ao controle negativo. Este resultado foi semelhante para todos os microrganismos estudados: Staphylococcus aureus (p=0,002), Candida tropicalis (p=0,002), Candida parapsilosis (p=0,001) e Candida albicans (p<0,001). Desta forma, apenas o Mavatricin® foi testado para o microrganismo arranjado em biofilme. Observou-se, então, que para a C.albicans e para o S. aureus, houve diferença significativa entre clorexidina e Malvatricin® (p<0,05), com melhores resultados para o clorexidina. Para C. tropicalis, o Malvatricin® diferiu significativamente (p<0,05) da água destilada. Em relação a C.parapsilosis, nenhuma diferença foi observada em relação ao controle negativo (p=0,468). Conclusão: Apesar de alguns fármacos alternativos serem tidos como antimicrobianos, tais propriedades sobre células planctônicas e, principalmente, sobre biofilme foram observadas apenas para o Malvatricin®.

Objective: To evaluate in vitro the antimicrobial potential of Anapyon®, Água Rabelo and Malvatricin® against oral microorganisms.Method: The experiment used a four-day sequential protocol that evaluated by optical density measurements the antimicrobial potential of these products against Staphylococcus aureus, Candida tropicalis, Candida parapsilosis and Candida albicans in their planktonic forms. Only Malvatricin® was also evaluated against microbial biofilms because it was the only one to produce satisfactory results against the planktonic forms. Distilled water was used as negative control and Chlorhexidine® as positive control. The data were analyzed statistically by the Kruskal-Wallis and Mann-Whitney tests. Results: According to the optical density readings, only Malvatricin® was effective against the planktonic microorganisms and its results were similar to those of the positive control (Chlorhexidine), which is the gold standard antimicrobial agent in dental research. Malvatricin® presented significantly higher efficacy than the other antiseptics (Anapyon® and Água Rabelo®) and the negative control, and this result was similar for all tested microorganisms: S. aureus (p=0.002), C tropicalis (p=0.002), C. parapsilosis (p=0.001) and C. albicans (p<0.001). For this reason, only Malvatricin® was evaluated against the microorganisms arranged as biofilms. Chlorhexidine presented significantly better results (p<0.05) than Malvatricin® against C. albicans and S. aureus. When tested against C. tropicalis, Malvatricin® differed significantly (p<0.05) from distilled water, while against C. parapsilosis no significant difference (p=0.468) was observed in comparison with the negative control. Conclusion: Although some pharmaceutical products being considered anti-microbial, such properties against planktonic cells and especially the biofilms were observed only for Malvatricin®.

Reconhecimento de Candida albicans por fibroblastos murinos: envolvimento de receptores de reconhecimento de patógenos (TLR2 e CD14) e a proteína adaptadora MyD88 / The recognition of Candida albicans by fibroblasts: Toll like receptors pathogens (TLR2 and CD14) abt the MyD88 protein evaluation

Os tecidos pulpar e periodontal são frequentemente agredidos por fatores ambientais como calor, trauma mecânico e micro-organismos, sendo estes considerados o fator etiológico principal das periodontopatias e periapicopatias. Dentre as células residentes desses tecidos, especial atenção tem sido dada ao papel dos fibroblastos no desenvolvimento da resposta imune. Fibroblastos são células que respondem à estímulos microbianos e existem evidências do papel de receptores do tipo Toll (TLR) no reconhecimento desses estímulos. Dessa forma, o presente trabalho teve como objetivo principal avaliar o reconhecimento de Candida albicans por fibroblastos gengivais e pulpares. Para tal, fibroblastos isolados a partir de tecido gengival e pulpar de camundongos do grupo controle e deficientes de TLR2, CD14 e MyD88 foram avaliados quanto à expressão de TLRs e moléculas de superfície, resposta proliferativa e produção de citocinas (TGF-β, IL-1β, TNF-α, IL-13 e IL-6), após a estimulação com agonistas de TLR2, TLR4 e C. albicans. Fibroblastos gengivais e pulpares, apesar de provenientes de tecidos diferentes, apresentaram características morfológicas semelhantes. Contudo, a cinética de crescimento dos fibroblastos gengivais deficientes de MyD88 foi mais lenta, e fibroblastos pulpares demoraram mais tempo para surgir a partir dos fragmentos de tecido. A ausência de TLR2 e da molécula adaptadora MyD88 não afetaram a produção de colágeno Tipo I pelos fibroblastos gengivais. Entretanto, fibroblastos deficientes de CD14 apresentaram baixa produção de colágeno. Ademais, os fibroblastos gengivais expressaram TLR2, TLR3, TLR4, assim como as moléculas de adesão ICAM-1 e CD44. A ausência de TLR2 e CD14 interferiu na resposta proliferativa de fibroblastos gengivais e pulpares, respectivamente. O reconhecimento de C. albicans por fibroblastos gengivais e pulpares modulou a produção das citocinas. A produção de TNF-α foi...

Pulpal and periapical tissue are frequently injured by heat, mechanical trauma and microorganisms, which are considered the main etiological factor of periodontal and endodontic diseases. Among these tissue resident cells, special attention has been given to fibroblasts in the immune response. Fibroblasts are cells that recognize pathogens through Toll like receptors (TLR). The aim of this study was to evaluate the recognition of Candida albicans by pulpal and gingival fibroblasts from TLR2, CD14, MyD88 knockout mice and control group mice. The results were analyzed concerning the expression of TLR(s) and surface molecules, proliferative response and citokynes production (TGF-β, IL-1β, TNF-α, IL-13 e IL-6) after the cells stimulation with TLR2, TLR4 and C.albicans agonists. Gingival and Pulpal fibroblasts, even isolated from different tissue, showed morphological similarities; however, gingival fibroblast deficient of MyD88 show lower proliferative response and pulpa l fibroblasts needed more time to detach from tissue fragments. The production of Type I collagen was affected in gingival cells deficient of CD14. Gingival fibroblasts expressed TLR2, TLR3, TLR4, and the adhesion molecules (ICAM-1 and CD44). The absence of TLR2 and CD14 interfered with the proliferative response of pulpal and gingival fibroblasts, respectively. The recognition of C. albicans by gingival and pulpal fibroblasts modulated the citokynes production. TNF-α production after the recognition of C. albicans was dependent from MyD88, CD14 and TLR2 molecules, whereas the production of IL-1β and IL-13 was dependent of TLR2.

Produção de citocinas pelos monócitos de idosos com estomatite protética associada a Candida / Cytokine production by monocytes from elderly patients with Candida-associated denture stomatitis

Estomatite protética associada a Cândida (EPC), a lesão mais frequente em usuários de próteses removíveis, principalmente os idosos, caracteriza-se por uma inflamação da mucosa bucal que suporta a prótese. Está fortemente associada com Candida Albicans, bem como com fatores locais e sistêmicos, como a deficiência da resposta imune. Os monócitos são importantes na resposta protetora contra o fungo, produzindo citocinas que recrutam e ativam leucócitos. Existem alterações funcionais dessas células com o avanço da idade. Não foi possível encontrar na literatura dados referentes à função imunomodulatória dos monócitos de idosos com EPC. O presente trabalho pretendeu avaliar a produção de citocinas por essas células, estimuladas in vitro com C. albicans, obtidas do sangue periférico de idosos usuários de prótese total superior (PTS) com EPC, comparando-se com idosos usuários de PTS sem EPC, e com idosos e jovens não usuários de PTS. Os monócitos isolados foram cultivados em placas de cultura de 24 poços, na ausência ou presença de lipopolissacarídeo (LPS) ou C. albicans ATCC 90028 morta pelo calor. Após 18 horas, o sobrenadante foi coletado e submetido ao ensaio de imunoabsorção por ligação enzimática (ELISA) para dosagem das citocinas pró- inflamatórias fator de necrose tumoral- (TNF-), interleucina-6 (IL-6), IL-1, CXCL8 e proteína quimiotática de monócito (MCP-1), e anti-inflamatórias IL-10 e fator transformador de crescimento- (TGF-). Os resultados estão expressos como média ± desvio padrão dos valores obtidos para cada grupo, e foram analisados por meio de testes estatísticos não-paramétricos. Valores de p<0,05 foram considerados estatisticamente significativos. Os resultados demonstraram, de uma forma geral, alterações nos monócitos oriundos dos idosos com EPC, em comparação aos outros grupos: menor produção espontânea de CXCL8 e de MCP-1; menores níveis de TNF-, de IL-6, de IL-1, de CXCL8, de MCP-1 e de IL-10, após estímulo com LPS; e menor...

Candida-associated denture stomatitis (DS), the most frequent lesion among denture wearers, especially the elderly, is characterized by inflammation of the denture-bearing mucosa. It is strongly associated with Candida albicans, as well as with local and systemic factors, such as impaired immune response. Monocytes are important in the protective immune response against the fungus, by the production of cytokines that recruit and activate leukocytes. There are functional changes of these cells with advancing age. No data were found in the literature concerning the immunomodulatory function of these phagocytes in elderly patients with DS. This study aimed to evaluate the cytokine production by monocytes, challenged in vitro with C. albicans, obtained from peripheral blood of elderly denture wearers with DS, compared with elderly denture wearers without DS and elderly and young non-denture wearers. The isolated monocytes were cultivated in 24-well flat-bottomed culture plates, in the absence or presence of lipopolysaccharide (LPS) or heat-killed C.albicans ATCC 90028. After 18 hours, the supernatant was collected and submitted to the enzyme-linked immunosorbent assay (ELISA) for determination of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-), interleukin-6 (IL-6), CXCL8, IL-1, monocyte chemotactic protein-1 (MCP-1) and anti-inflammatory cytokines IL-10 and transforming growth factor- (TGF-). The results are expressed as mean ± standard deviation of the values obtained for each group, and were analyzed using nonparametric statistical tests; p values <0.05 were considered statistically significant. The results demonstrated, in general, changes in monocytes from the elderly with DS, as compared to other groups: lower spontaneous production of CXCL8 and MCP-1; lower levels of TNF-, IL-6, IL-1, CXCL8, MCP-1 and IL-10 after stimulation with LPS; and reduced production of TNF- and IL-6 after stimulation with C. albicans. Comparing...

Effect of shark liver oil on peritoneal murine macrophages in responses to killed-Candida albicans

Shark Liver Oil [SLO] is an immunomodulator. Macrophages play a key role in host defense against pathogens like fungi. Candida albicans have mechanisms to escape immune system. We determined the effect of killed-Candida on the in vitro viability of macrophages and the effect of SLO on augmentation of this potency. Peritoneal macrophages were separated and cultured [3-105/well]. At first, the effect of killed-Candida [200 cells/well] on macrophage viability was evaluated, using MTT test. Then, MTT was performed on macrophages stimulated with killed-Candida in the presence of SLO. Killed-Candida suppressed the ability of MTT reduction and hence macrophages viability [P=0.026], but addition of SLO [100 mg/ml] significantly enhanced cell viability [P=0.00]. So, SLO could neutralize the inhibitory effect of Candida. Simultaneous with cytotoxic effect of killed-Candida cells on macrophages viability, SLO augment macrophages viability. So, it can be applied in candidiasis as a complement

Effect of Allicin of garlic on production nitric oxide of macrophage to Candida albicans

Garlic is an herbal plant having various compounds. one of the most important of which is allicin with antibiotic property. Candida Albicans is opportunistic yeast which in case of immune system dysfunction is considered as a pathogenic agent. Immune mechanisms against this fungus are Macrophages which act with oxidative and non-oxidative fungicide mechanisms. Oxidative mechanism includes active oxygen and nitrogen mediators which are produced by active macrophages and thereby the microorganisms are eliminated. The aim of this study was to investigate Allicin effects on the increase activites macrophage to product of Nitric Oxide It is examined in this study the effect of garlic allicin on macrophages' activity in releasing Nitric oxide against Candida Albicans. Garlic allicin was prepared by the method of chloroformic extract, and then made to react with macrophages from male mouse Balb/c with 2-8 week of age in vitro. Candida Albicans was divided into two groups: with and without allicin. Both cases compared as positive and negative samples, the rate of macrophages' activity was determined through production of Nitric Oxide. After examining the rate of Nitric Oxide produced by macrophages, results showed that allicin as a natural material activates immune system against this fungus, so that macrophages with allicin can produce more Nitric Oxide than the group without allicin. This was obtained by comparing the results from these two groups and the control group. Regarding the important role of Candida Albicans in Candidiasis and applying the preventing agents by this fungus in suppressing the macrophages' activities in producing Nitric Oxide, and also by studying related literatures, we concluded that allicin extracted from garlic can affect greatly on the macrophages' activities in producing Nitric Oxide against agents of Candidiasis disease

Ação dos leucotrienos na fagocitose via receptores Fc`GAMA´ e manose em macrófagos alveolares e mecanismos moleculares envolvidos / Importance of leukotrienes en phagocytosis of Fc`GAMA´ and mannose receptor in alveolar macrophages and molecular mecanismes involved

Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB`IND.4´ e LTD`IND.4´ aumentam a fagocitose via Fc`GAMA´R de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da Pi3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via Fc`GAMA´R e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB`IND.4´ envolveu predominantemente a via ERK1/2 e PKC`ALFA´ e com menor intensidade da PKC`DELTA´...

Heterologous expression and biochemical characterization of an alpha 1, 2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.

Recurring Candida albicans esophagitis in a HIV-infected patient undergoing long-Term antiretroviral therapy, and with absent-negligible immunodeficiency

A patient with HIV infection developed the first episode of AIDS-defining opportunism (severe Candida albicans esophagitis) with an underlying CD4+ lymphocyte count of 1,025 cells/µL. After treatment with a highly active antiretroviral therapy (HAART), taken with insufficient compliance and leaving a residual viral load, our patient suffered from two relapses of esophageal candidiasis, which occurred three months and seven years later, when his CD4+ lymphocyte count was 930 and 439 cells/µL, respectively, and a viral load slightly above 10(4) copies/mL was still present. Also in the HAART era, Candida esophagitis remains one of the most common AIDS-defining diseases, but a presentation with a concurrent CD4+ count above 1,000 cells/µL remains a rare exception, as well as the two isolated, subsequent relapses, occurred with a CD4+ count ranging from 439 to 930 cells/µL, and a residual HIV viremia due to insufficient adherence to the prescribed HAART regimens. Our case report represents the opportunity to revisit the epidemiology and, especially, the pathogenesis of this opportunistic fungal complication in HIV-infected patients and in other subjects at risk, on the ground of an extensive literature review, and to explore possible alternative supporting factors other than the crude absolute CD4+ lymphocyte count, with emphasis on the possible role of a persisting HIV viremia, and other potential contributing factors. Clinicians engaged with immunocompromised patients and subjects with HIV disease, should be aware that a Candida esophagitis may occur and relapse also when the cell-mediated immunity, as measured by a simple CD4+ cell count, do not show relevant abnormalities.

The use of MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei.

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

Three suitable antigens for delayed-type hypersensitivity skin testing in a tropical country like Thailand.

Delayed type hypersensitivity (DTH) skin test is a standard tool to assess in vivo cell-mediated immunity. Mantoux method using 4-5 common recalled antigens is recommended. However, not all antigens are widely available and appropriate antigens for tropical countries are not known. The objective of this study is to investigate what and how many antigens should be included in the DTH testing panel that suitable for Thailand and may be for this region. The DTH skin tests were done by Mantoux method in a double blinded fashion. Average induration size of > or = 5 mm defined as a positive test. Antigens included purified protein derivative (PPD), Candida albicans, tetanus toxoid (TT), Trichophyton mentagrophytes and hepatitis B vaccine (HBV). The negative control was normal saline. Of 95 healthy subjects, all showed DTH positive to > or = 1 antigen. The positivity to C. albicans, tetanus toxoid, PPD, T. mentagrophytes, and HBV was 92.6%, 83.2%, 82.1%, 50.5%, and 5.3%, respectively. When three antigens: PPD, TT and C. albicans were analyzed, 100% of subjects showed a positive response to > or = 1 antigen and 96.8% showed a positive response to > or = 2 antigens. When only PPD and TT were analyzed, 100% of subjects showed > or = 1 antigen positive and 68.4% showed both antigens positive. C. albicans antigen at 1:100 was associated with a high incidence of fever (2/20) and large local reaction (7/20), 1:500 was found to be the optimal concentration. PPD, TT and C. albicans are suitable to be included in a DTH skin testing in a tropical country like Thailand. However, in a setting where C. albicans extract is not available, testing with only two antigens of PPD and tetanus toxoid may be an alternative, but with a lower sensitivity.

Delayed hypersensitivity skin testing in the Thai adult population.

OBJECTIVE: The present study was carried out in healthy Thai subjects to determine the types and concentrations of standard antigens used in delayed-type hypersensitivity (DTH) skin testing as an evaluation of cell-mediated immunity. MATERIAL AND METHOD: One hundred subjects were tested with three antigens including tuberculin (purified protein derivative), tetanus toxoid, 1:10 and 1:100 dilutions, and Candida albicans, 1:10 and 1:100 dilutions. RESULTS: We found that 92% of the subjects responded to tuberculin and/or tetanus toxoid at a 1:10 dilution, 77% responded to tetanus toxoid at a 1:10 dilution, and 35% responded to a 1:100 dilution. There was only one subject who responded to Candida albicans at a 1:10 dilution, and none to a 1:100 dilution. The size of tuberculin reactions varied from 5 mm to over 20 mm without any evidence of active tuberculosis. CONCLUSION: Tuberculin and tetanus toxoid at a 1:10 dilution are probably sufficient to be used in DTH skin testing to evaluate T-cell function.

Candida spp en pacientes VIH/SIDA / Candida spp in patient VIH/SIDA

La candidiasis es la infección fúngica más frecuente en pacientes VIH seropositivos y con SIDA, siendo C. albicans la especie que predomina. En estos pacientes, la candidiasis se manifiesta más comúnmente como orofaringitis, con una incidencia que oscila entre 45 por ciento y 95 por ciento de todos los casos. En Venezuela son pocos los reportes que existen acerca de esta entidad en pacientes VIH/SIDA, por lo cual uno de los objetivos de esta investigación fue evaluar este aspecto. Se estudió una cohorte de 402 pacientes con VIH/SIDA con o sin orofaringitis y con o sin tratamiento antifúngico. Las especies de Candida más importantes aisladas fueron: C. albicans (80,41 por ciento), C. tropicalis (8,78 por ciento), C. glabrata (4,90 por ciento), C. dubliniensis (3,26 por ciento). Se evaluó la susceptibilidad de los diferentes aislados a las drogas antifúngicas con el método de la NCCLS y el método de los pozos de difusión. El 37,5 por ciento C. albicans provenientes de pacientes con tratamiento antimicótico fueron resistentes al fluconazol (resistencia secundaria) y 23,95 por ciento mostraron también resistencia al itraconazol (resistencia cruzada). Los ailados obtenidos se tipificaron a través de la reacción en cadena de la polimera (RAPD) y se estableció la que la misma representa una técnica útil para diferenciar Candida spp. En esta cohorte de pacientes evaluados, las características clínicas y demográficas son similares a las reportadas mundialmente, y que desde la introducción del TARGA, la frecuencia de las infecciones por Candida spp disminuyó. Hasta la presente fecha, en Venezuela no se había reportado el hallazgo de C. dubliniensis (3,26 por ciento).