Aplicação do plasma de baixa temperatura sob pressão atmosférica como adjuvante ao tratamento da mucosite oral / Application of low temperature atmospheric pressure plasma as an adjuvant in treatment oral mucositis

A mucosite oral é um quadro clínico que acomete frequentemente pacientes sob terapia antineoplásica na região de cabeça e pescoço e caracteriza-se por ulcerações na mucosa que geram intensa dor local, odinofagia, aumento do risco de infecções, do uso de antibióticos e do tempo de hospitalização. A correlação entre mucosite oral, infecção fúngica e o potencial de disseminação fúngica sistêmica foi recentemente descrita. Apesar do impacto desse quadro clínico sobre a qualidade e tempo de vida dos pacientes oncológicos, não há consenso sobre a profilaxia e o protocolo terapêutico. O plasma de baixa temperatura sobre pressão atmosférica (LTAPP) apresenta efeito antimicrobiano, anti-inflamatório e reparador tecidual, o que sugere que possa ser promissor no tratamento da mucosite oral. Os objetivos gerais deste projeto foram divididos em dois subprojetos: 1) Definir os melhores parâmetros in vitro com efeito antifúngico e não tóxico e avaliar o LTAPP no tratamento de lesão de mucosite oral em modelo murino de mucosite por quimioterapia e 2) avaliar se o tratamento com LTAPP pode prevenir a disseminação fúngica sistêmica em ratos a partir de infecção experimental de lesões de mucosite oral por Candida albicans. Para tal, foram incluídos no estudo 100 ratos (Rattus norvegicus) com 90 a 100 dias de idade. No subprojeto 1, a lesão de mucosite oral foi induzida por administração de 5 fluorouracila (5-FU), enquanto no subprojeto 2, utilizou-se 5-FU associada à cisplatina ambas associadas à aplicação tópica de ácido acético 50%. Para o subprojeto 1, os animais foram randomicamente divididos em 2 grupos experimentais (n=30): a) Grupo mucosite; b) Grupo mucosite tratado com LTAPP, avaliados após 1, 5 e 12 dias do tratamento. Durante o período experimental, as lesões foram fotografadas e a gravidade da mucosite classificada por meio da atribuição de escores. Após a eutanásia e o processamento, os cortes histológicos corados por hematoxilina-eosina (HE) foram analisados microscopicamente. Para o subprojeto 2, o estudo de disseminação sistêmica fúngica nos grupos de mucosite infectada com C. albicans tratado ou não com LTAPP foi conduzido pelo isolamento fúngico a partir de amostras de sangue total e macerado dos órgãos. Para tanto foram estudados 2 grupos de ratos (n=20): c) Grupo mucosite infectado com C. albicans e d) Grupo mucosite infectado com C. albicans tratada com LTAPP, avaliados após 24 e 72 h do tratamento. Para ambos os projetos, o melhor parâmetro in vitro foi selecionado, isto é aquele com maior atividade antifúngica e baixa toxicidade. Dessa forma, as lesões foram expostas ao LTAPP de hélio por 5 min na distância de 1,5 cm na potência de 1 W. Os resultados in vitro mostraram que o LTAPP teve efeito antifúngico e baixa toxicidade para células de mamíferos. Os resultados in vivo mostraram que 5-FU afetou a saúde geral dos animais, evidenciada pela perda de peso corporal. Em ambos os grupos, houve reparo tecidual após 12 dias do tratamento, com resolução quase completa da lesão, o que foi corroborado pelos achados microscópicos. O grupo LTAPP exibiu uma tendência maior de redução da lesão, após 12 dias de tratamento. Além disso, o LTAPP apresentou efeito inibitório sobre C. albicans após 5 minutos, de exposição, com redução da recuperação fúngica da língua após 24 h (p<0.05). A disseminação fúngica sistêmica foi reduzida significativamente após 24 e 72 h do tratamento. Com base nos resultados obtidos, conclui-se que o LTAPP é uma ferramenta promissora para futura aplicação clínica em pacientes com mucosite oral. (AU)

Oral mucositis is a clinical condition that frequently affects patients undergoing antineoplastic therapy in the head and neck region and is characterized by mucosal ulcerations that generate intense local pain, odynophagia, increased risks of infections, use of antibiotics and the length of hospital stay. The correlation among oral mucositis, fungal infection and the potential for systemic fungal dissemination has recently been described. Despite the impact of this clinical condition on the quality and life expectancy of cancer patients, there is no consensus on prophylaxis and the therapeutic protocols. Low temperature atmospheric pressure plasma (LTAPP) has antimicrobial, anti-inflammatory and tissue repairing effects, which suggests that it can be promising in the treatment of oral mucositis. The general objectives of this project were divided into two subprojects: 1) Define the best antifungal and non-toxic in vitro parameters and to evaluate the application of LTAPP in the treatment of oral mucositis in murine model for chemotherapy, and 2) to evaluate whether treatment with LTAPP can prevent systemic fungal dissemination in rats from experimental infection of oral mucositis lesions by Candida albicans. A total of 100 rats (Rattus norvegicus) aged 90 to 100 days were included in the study. In subproject 1, oral mucositis lesion was induced by administration of only 5- fluorouracil (5-FU), while in subproject 2, administration and systemic administration of 5-FU associated with cisplatin, both associated with topical application of 50% acetic acid. For subproject 1, the animals were randomly divided into 2 experimental groups (n=30):a) Mucositis group and b) Mucositis group treated with LTAPP evaluated after 1, 5 and 12 days of treatment. During the experimental period, the lesions were photographed, and the severity of mucositis was classified into scores. After euthanasia and processing, the histological cuts stained by hematoxylin-eosin (HE) were analyzed. For subproject 2, the study of fungal systemic dissemination in groups of mucositis infected with C. albicans treated or not with LTAPP was conducted by fungal isolation from whole blood and macerated organs. Therefore, 2 groups of rats (n=20) were studied: c) Mucositis group infected with C. albicans and d) Mucositis group infected with C. albicans treated with LTAPP, evaluated after 24 and 72 h of treatment. For both subprojects, the best in vitro parameter was selected, that is, the one with the greatest antifungal effect and low toxicity. Thus, the lesions were exposed to helium LTAPP for 5 min at a distance of 1.5 cm at power of 1 W. In vitro results showed that LTAPP has an antifungal effect and low toxicity. In vivo results showed that 5-FU affected the general health of animals evidenced by body weight loss. In both groups, there was tissue repair after 12 days of treatment, with almost complete resolution of the lesion, which was corroborated by the microscopic findings. LTAPP group showed a greater trend of reduction of lesion, after 12 days of the treatment. Furthermore, LTAPP showed inhibitory effect on C. albicans after 5 min of exposition, with reduction in fungal recovery from the tongue after 24 h (p<0.05). Reduction in fungal dissemination was observed after 24 and 72 h of LTAPP treatment (p<0.05). Based on the obtained results, it was concluded that LTAPP is a promising tool for future clinical application in patients with oral mucositis.(AU)

Effect of the pH on the Antibacterial Potential and Cytotoxicity of Different Plasma-Activated Liquids.

In this study, different plasma-activated liquids were evaluated for their antimicrobial effects against Escherichia coli, as well as for their cytotoxicity on mammalian cells. The PALs were prepared from distilled (DIS), deionized (DI), filtered (FIL), and tap (TAP) water. Additionally, 0.9% NaCl saline solution (SAL) was plasma-activated. These PALs were prepared using 5 L/min air gliding arc plasma jet for up to 60.0 min of exposure. Subsequently, the physicochemical properties, such as, the oxidation-reduction potential (ORP), the pH, the conductivity, and the total dissolved solids (TDS) were characterized by a water multiparameter. The PALs obtained showed a drastic decrease in the pH with increasing plasma exposure time, in contrast, the conductivity and TDS increased. In a general trend, the UV-vis analyses identified a higher production of the following reactive species of nitrogen and oxygen (RONS), HNO2, H2O2, NO3-, and NO2-. Except for the plasma-activated filtered water (PAW-FIL), where there was a change in the position of NO2- and NO3- at some pHs, The higher production of HNO2 and H2O2-reactive species was observed at a low pH. Finally, the standardized suspensions of Escherichia coli were exposed to PAL for up to 60.0 min. The plasma-activated deionized water (PAW-DI pH 2.5), plasma-activated distilled water (PAW-DIS pH 2.5 and 3), and plasma-activated tap water (PAW-TAP 3.5) showed the best antimicrobial effects at exposure times of 3.0, 10.0, and 30.0 min, respectively. The MTT analysis demonstrated low toxicity of all of the PAL samples. Our results indicate that the plasma activation of different liquids using the gliding arc system can generate specific physicochemical conditions that produce excellent antibacterial effects for E. coli with a safe application, thus bringing future contributions to creating new antimicrobial protocols.

Silver-Coated Silica Nanoparticles Modified with MPS: Potential Antimicrobial Biomaterials Applied in Glaze and Soft Reliner.

Soft reliner and glaze are materials used over full or partial dental prosthesis to prevent excessive pressure on the supporting tissues. They are also indicated as supportive treatment for dental stomatitis, especially when modified by the addition of medications. The objective of the work was to evaluate the antimicrobial effect of silver-coated silica nanoparticles in a glaze and a soft reliner. The nanoparticles were synthesized, characterized, and tested by minimum inhibitory concentration (MIC) for C. albicans SC5314. Then, the nanoparticles were incorporated to a glaze and a soft reliner, which were called nanocomposites. Then, the nanocomposites were divided into six groups (n = 12): CG: glaze/reliner; CR: reliner; G1: glaze + 1% nanoparticles/reliner; G2: glaze + 2.5% nanoparticles/reliner; R1: reliner + 1%; R2: reliner + 2.5%. The nanocomposites were characterized by a goniometer and by a scanning electron microscope. The antibiofilm test was performed against C. albicans SC5314. According to the MIC results, the non-functionalized nanoparticles reduced fungal growth at 1000 µg/mL and the functionalized nanoparticles at 2000 µg/mL. The functionalized nanoparticle had a superior dispersion being selected for the antibiofilm test. There was a reduction of 64% in CFU/specimen count for the glaze, not statistically significant (p = 0.244). For the soft reliner, there was an increase in CFU/specimen with the presence of nanoparticles, still not statistically significant (p = 0.264). In conclusion, it is necessary to conduct new studies to increase the release of silver, thus improving nanoparticles' antifungal potential.

Applications of Plasma-Activated Water in Dentistry: A Review.

The activation of water by non-thermal plasma creates a liquid with active constituents referred to as plasma-activated water (PAW). Due to its active constituents, PAW may play an important role in different fields, such as agriculture, the food industry and healthcare. Plasma liquid technology has received attention in recent years due to its versatility and good potential, mainly focused on different health care purposes. This interest has extended to dentistry, since the use of a plasma-liquid technology could bring clinical advantages, compared to direct application of non-thermal atmospheric pressure plasmas (NTAPPs). The aim of this paper is to discuss the applicability of PAW in different areas of dentistry, according to the published literature about NTAPPs and plasma-liquid technology. The direct and indirect application of NTAPPs are presented in the introduction. Posteriorly, the main reactors for generating PAW and its active constituents with a role in biomedical applications are specified, followed by a section that discusses, in detail, the use of PAW as a tool for different oral diseases.

Effect of Cold Atmospheric Plasma Jet Associated to Polyene Antifungals on Candida albicans Biofilms.

The increasing incidence of antifungal resistance represents a great challenge in the medical area and, for this reason, new therapeutic alternatives for the treatment of fungal infections are urgently required. Cold atmospheric plasma (CAP) has been proposed as a promising alternative technique for the treatment of superficial candidiasis, with inhibitory effect both in vitro and in vivo. However, little is known on the association of CAP with conventional antifungals. The aim of this study was to evaluate the effects of the association between CAP and conventional polyene antifungals on Candida albicans biofilms. C. albicans SC 5314 and a clinical isolate were used to grow 24 or 48 h biofilms, under standardized conditions. After that, the biofilms were exposed to nystatin, amphotericin B and CAP, separately or in combination. Different concentrations of the antifungals and sequences of treatment were evaluated to establish the most effective protocol. Biofilms viability after the treatments was compared to negative control. Data were compared by One-way ANOVA and post hoc Tukey (5%). The results demonstrate that 5 min exposure to CAP showed more effective antifungal effect on biofilms when compared to nystatin and amphotericin B. Additionally, it was detected that CAP showed similar (but smaller in magnitude) effects when applied in association with nystatin and amphotericin B at 40 µg/mL and 60 µg/mL. Therefore, it can be concluded that the application of CAP alone was more effective against C. albicans biofilms than in combination with conventional polyene antifungal agents.

Ellagic Acid-Cyclodextrin Complexes for the Treatment of Oral Candidiasis.

The increase in the prevalence of fungal infections worldwide and the rise in the occurrence of antifungal resistance suggest that new research to discover antifungal molecules is needed. The aim of this study was to evaluate the potential use of ellagic acid-cyclodextrin complexes (EA/HP-ß-CD) for the treatment of oral candidiasis. First, the effect of EA/HP-ß-CD on C. albicans planktonic cells and biofilms was evaluated. Then, the cytotoxicity of the effective concentration was studied to ensure safety of in vivo testing. Finally, the in vivo effectiveness was determined by using a murine model of induced oral candidiasis. Data was statistically analyzed. The minimal inhibitory concentration of EA/HP-ß-CD was 25 µg/mL and a concentration of 10 times MIC (250 µg/mL) showed an inhibitory effect on C. albicans 48 h-biofilms. The complex at concentration 250 µg/mL was classified as slightly cytotoxic. In vivo experiments showed a reduction in fungal epithelial invasion after treatment with EA/HP-ß-CD for 24 h and 96 h when compared to the negative control. In conclusion, the results demonstrated that EA/HP-ß-CD has antifungal and anti-inflammatory effects, reducing the invasive capacity of C. albicans, which suggests that EA/HP-ß-CD may be a promising alternative for the treatment of oral candidiasis.

Understanding SARS CoV-2 biology to win COVID-19 battle / Entendendo a biologia do SARS CoV-2 para vencer a batalha contra a COVID-19

This review highlights the main findings on the biology of SARS CoV-2 and the strategies to combat COVID 19 pandemic. Since the initial outbreak in China on December 2019, the international scientific community joined efforts in an unprecedent public health battle. In late May 2020, 5 204 508 cases and 337 687 deaths have been reported by World Health Organization, with higher number of cases in Europe and Americas. SARS-CoV-2 was described as a novel variant from the coronavirus family and its genome was sequenced within a few months while COVID 19 quickly spread worldwide. The main cell receptor (angiotensin converting enzyme 2) was identified as involved in the invasion of host cells. As a result of the findings from cell biology, immunology and clinical studies, the pathogenesis of the virus started to be understood but it has been not fully elucidated so far. While a massive effort for the development of a vaccine is on course, preventive protocols for infection control have been proposed. Many studies on the discovering of effective therapeutic protocols have been developed, particularly on the redirection of already approved substances, but no gold standard treatment was established until now. An overview on the envisioned socioeconomic and politic impacts suggest that our society will be transformed after COVID 19 pandemia. As a result, deep changes in science, politics, socioeconomic and healthcare priorities shall appear in post-pandemia agenda.(AU)

Esta revisão destaca os principais relatos sobre a biologia do SARS CoV-2 e as estratégias para combater a epidemia de COVID 19. Desde o surto inicial na China, em dezembro de 2019, a comunidade científica internacional uniu esforços em uma batalha de saúde pública sem precedentes. No final de maio de 2020, 5.204.508 casos e 337.687 mortes foram reportadas pela Organização Mundial da Saúde, com maior número de casos na Europa e nas Américas. O SARS-CoV-2 foi descrito como uma nova variante da família coronavírus e seu genoma foi sequenciado em poucos meses, enquanto a COVID 19 se espalhou rapidamente pelo mundo. O receptor celular principal (angiotensin converting enzyme 2) foi identificado como envolvido no processo de invasão às células do hospedeiro. Como resultado das descobertas da biologia celular, imunologia e estudos clínicos, a patogênese do vírus começou a ser entendida mas não está completamente elucidada até o momento. Enquanto um grande esforço para o desenvolvimento da vacina está em curso, protocolos preventivos para o controle de infeção foram propostos. Muitos estudos para o estabelecimento de protocolos terapêuticos efetivos têm sido desenvolvidos, particularmente no reposicionamento de substâncias já aprovadas, porém nenhum tratamento padrão foi estabelecido até o momento. Uma visão geral dos impactos políticos e socioeconômicos previstos sugerem que nossa sociedade será transformada após a pandemia de COVID 19. Como resultado, mudanças profundas nas prioridades da ciência, política, área socioeconômica e saúde deverão surgir na agenda póspandemia.(AU)

Avaliação do potencial de aplicação do ácido elágico nanoencapsulado em ciclodextrina no tratamento de candidose bucal / Evaluation of the potential application of cyclodextrin nanoencapsulated ellagic acid for the treatment of oral candidosis

O aumento crescente da prevalência das infecções fúngicas no cenário mundial e aumento da ocorrência de resistência aos antifúngicos convencionais sugerem que pesquisas para a descoberta de novas moléculas antifúngicas são urgentemente necessárias. O objetivo geral deste estudo é contribuir para a prospecção de alternativas terapêuticas, avaliando nanoencapsulados da substância bioativa ácido elágico (NAE) no tratamento da candidose bucal. Os seguintes objetivos específicos foram: complexar ácido elágico em ciclodextrina, em concentração com atividade antifúngica frente C. albicans; caracterizar quimicamente o encapsulado; prospectar possíveis mecanismos de ação; avaliar ação de concentrações subinibitórias sobre a produção de exoenzimas e aderência às células epiteliais; avaliar citotoxicidade do NAE nas condições de tratamento efetivas in vitro e in vivo (Drosophila melanogaster) e avaliar ação no tratamento in vivo de lesões de candidose oral induzida em modelo murino. Os dados foram analisados estatisticamente, adotando-se significância de 5%. HP-ß-CD formou complexos solúveis de inclusão com AE. A porcentagem de AE/HP-ß-CD foi de 15,25 ± 0,49%. As análises de MEV e FTIR confirmaram a formação de complexo de inclusão. AE/HP-ß-CD manteve a atividade antifúngica do AE puro, com CIM 25 µg/ml para C. albicans ATCC 18804 e 50 µg/ml para C. albicans SC 5314. O tratamento de C. albicans com sub-CIMs indicou aumento da CIM na presença de sorbitol para ATCC 18804 (50 µg/ml), sugerindo ação sobre parede celular. Os resultados sugerem que não há ação sobre membrana celular. Não foi detectado efeito sobre morfogênese e produção de enzimas extracelulares. Efeito protetor de AE a 3,2, 6,4 e 32 µg/ml foi observado em modelo D. melanogaster infectado com C. albicans que resultou na sobrevivência de 45, 33 e 34%. Baixa toxicidade foi observada nas concentrações de 200 e 250 µg/ml. No modelo de candidose bucal em camundongos foi observada redução significativa da invasão de hifas no epitélio no grupo tratado AE/HP-ß-CD em relação aos controles, em 24 horas e 48 horas. Conclui-se que AE/HP-ß-CD apresentou efeito inibitório sobre C. albicans in vitro, com baixa toxicidade. Em modelo Drosophila melanogaster apresentou efeito protetor frente à infecção fúngica. Em modelo murino levou à redução na invasão epitelial pelo fungo(AU)

The increase in the prevalence of fungal infections worldwide and the rise in the occurrence of antifungal resistance suggest that new researches for the discovery of antifungal molecules are needed. The aim of this study is to contribute to the prospection of therapeutic alternatives, evaluating nanoencapsulates of ellagic acid (NAE) for the treatment of oral candidosis. In order to reach the general aim, the following specific objectives were determined: to complex the ellagic acid in cyclodextrin at effective concentration against C. albicans; to chemically characterize the encapsulate; to test the activity of the encapsulate using an invasive candidosis in vitro model, searching for the mechanisms of action; to evaluate the activity of subinhibitory concentrations on the exoenzymes production, and adherence to epithelial cells; to evaluate the cytotoxicity of NAE in the effective treatment conditions in vitro and in vivo (Drosophila melanogaster); and to evaluate the in vivo effectiveness for the treatment of oral candidosis in murine model. The obtained data was statistically analyzed (level of significance 5%). HP-ß-CD formed soluble inclusion complexes with EA. The percentage of EA/HP-ß-CD was 15.25 ± 0.49%. SEM and FTIR analyses confirmed the formation of inclusion complex. AE/HP-ß-CD showed the same antifungal activity of pure EA with MIC value of 25 µg/ml for C. albicans ATCC 18804 and 50 µg/ml for C. albicans SC 5314. Treatment with sub-MIC on C. albicans revealed increased MIC value in the presence of sorbitol for ATCC 18804 (50 µg/ml), suggesting activity on cell wall. Results suggest no effect on cell membrane. No effects of EA on morphogenesis and production of extracellular enzymes were detected. Protective effect of EA at 3.2, 6.4 e 32 µg/ml was observed in D. melanogaster model, resulting in survivals of 45, 33 and 34%. Low cytotoxicity was observed for concentrations of 200 and 250 µg/ml. In vivo tests in murine models indicated reduction in epithelial invasion after treatment EA/HP-ß-CD for 24 and 48 h when compared to control. In conclusion, EA/HP-ß-CD showed inhibitory effect on C. albicans in vitro, with low toxicity. Protective effect against fungal infection was observed in D. melanogaster model. In murine model of oral candidosis, EA/HP-ß-CD reduced fungal epithelial invasion(AU)

In Vivo Efficacy of Ellagic Acid against Candida albicans in a Drosophila melanogaster Infection Model.

The aim of this study was to evaluate the antifungal activity and the toxicity of ellagic acid (EA) using a Drosophila melanogaster model. Candida albicans bacteria were inoculated into Toll heterozygous flies. Survival curves were obtained for the evaluation of the antimicrobial effect and toxicity of EA. A protective effect of EA against fungal infection in Drosophila melanogaster was observed at nontoxic concentrations. This study showed that EA is a promising tool for the treatment of candidiasis.