Effects of rosmarinic acid and doxorubicine on an ovarian adenocarsinoma cell line (OVCAR3) via the EGFR pathway

Purpose: We aimed to reveal the effects of rosmarinic acid (RA), which has come to the forefront with its antitumor and antioxidant properties in many studies recently in the ovarian adenocarcinoma cell line, on the epidermal growth factor receptor (EFGR) signaling pathway in the presence of doxorubicin (DOX). Methods: Ovarian adenocarcinoma cell line (OVCAR3) and human skin keratinocyte cell line human skin keratinocyte cell line (HaCaT) were used as control. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied to determine the effect of RA and DOX on the proliferation of OVCAR3 and HaCaT cells. Bcl2 expression and epidermal growth factor receptor (EGFR) and western blot analysis were performed to determine the expression levels of the markers. Results: It was determined that RA (IC50 = 437.6 µM) and DOX (IC50 = 0.08 µM) have the ability to inhibit the proliferation of OVCAR3 cells and induce apoptosis in a 72-hour time and dose-dependent manner. Western blot showed that the expression level of Bcl-2 and EGFR in OVCAR3 cells was down-regulated by RA and DOX. Conclusions: Apoptosis in OVCAR3 cells can potentially be induced by RA via the EGFR pathway, and RA may be a potent agent for cancer therapy.

Desarrollo y validación de un método de suspensión basado en células Vero para la detección del virus Zika / Development and validation of a vero cell-based suspension method for the detection of Zika virus

Objetivo . Desarrollar y validar un método de suspensión celular utilizando células Vero 76 para el cultivo del virus Zika (ZIKV) basado en la infección de células recién sembradas no adheridas. Material y métodos . Se utilizaron tres multiplicidades de infección diferentes del ZIKV para desarrollar y comparar este novedoso método con el método estándar de monocapa de células confluentes. Además, validamos preliminarmente el método de suspensión utilizando muestras clínicas caracterizadas como positivas o negativas para el ZIKV. El método estándar de monocapa se utilizó como método de referencia, y el aislamiento viral se confirmó mediante un RT-PCR específico del ZIKV. Se estimó la sensibilidad e intervalos de confianza del 95% para el método de suspensión. Asimismo, se realizó una comparación técnica del método de suspensión contra el método de monocapa. Resultados . Nuestros hallazgos sugieren que tanto la carga viral como la replicación del ZIKV fueron comparables entre los métodos de infección en monocapa y en suspensión. Aunque ambos métodos fueron adecuados para cultivar y aislar el ZIKV, el método de suspensión se caracterizó por ser más fácil, barato y rápido, así como una técnica de aislamiento sensible. En comparación con el método de monocapa, el método de suspensión fue cuatro veces más sensible en la detección del ZIKV en casos inconclusos por RT-PCR. Conclusiones . El método de suspensión tiene el potencial de ser un método eficaz para cultivar y aislar el ZIKV y su uso es potencialmente útil tanto en la investigación como en entornos clínicos.

Objective. To develop and validate a cell suspension method using Vero 76 cells for culturing Zika virus (ZIKV) based on infection of detached freshly seeded cells. Material and methods. Three different multiplicities of infection of ZIKV were used to develop and compare this novel method to the standard confluent cell monolayer method. In addition, we preliminary validated the cell suspension method using well-characterized ZIKV positive and negative clinical samples. The standard confluent cell monolayer method was used as the reference method, and viral isolation was confirmed by a ZIKV-specific RT-PCR. The sensitivity and its 95% confidence intervals for the cell suspension method were estimated. Also, a technical comparison of the cell suspension method against the cell monolayer method was performed. Results. Our findings suggested that both the viral load and replication of ZIKV were comparable between both monolayer- and suspension-infection methods. Although both methods were suitable for culturing and isolating ZIKV, the cell suspension method was easier, cheaper, and quicker as well as a sensitive isolation technique. The cell suspension method was significantly more sensitive in detecting Zika in inconclusive cases by RT-PCR, with a fourfold increase compared to the confluent cell monolayer method. Conclusion. The cell suspension method has the potential to be an effective method for cultivating and isolating ZIKV and its application is potentially useful in both research and clinical settings.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Devascularized bone surface culture for identification of microorganisms for osteomyelitis / 中华创伤骨科杂志

Objective:To evaluate devascularized bone surface culture for identification of microorganisms for osteomyelitis.Methods:A prospective study was conducted to include the eligible patients who were diagnosed with osteomyelitis and treated at Division of Orthopaedics and Traumatology, Department of Orthopaedics and Traumatology, Nanfang Hospital from December 2021 to January 2023. Their infected bone tissues were collected for both bone sample culture (BSC) and general sample culture (GSC). For BSC, the devascularized bone fragments, harvested intraoperatively, were put flat on sterile culture plates with solidified agar, their surface was gently covered with cooled and molten tryptone soy agar, and then the plates with bone samples were incubated at 37 ℃ with 5% CO 2. Meanwhile, 5 suspected samples of infected bone tissue were randomly harvested by 5 independent instruments for laboratory GSC. The culture time, bacterial species, and bacterial positive rate were compared between the 2 culture methods. Results:Included were a total of 73 patients [59 males and 14 females with an age of 49.0(31.0, 58.5) years]. The culture time for BSC [1 (1, 1) d] was significantly shorter than that for GSC [3 (2, 3) d], and the total positive rate of BSC [78.1% (57/73)] was significantly higher than that of GSC [61.6% (45/73)] ( P<0.05). The bacterial species cultured by GSC were consistent with those cultured by BSC. Conclusion:In identification of microorganisms for osteomyelitis, since BSC may be quicker and lead to a higher positive rate of bacterial culture than GSC, it can be used as an optional choice besides GCS.

Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow

ABSTRACT Introduction: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12μM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

Ovarian Tissue Culture to Preserve Fertility in Transgender Male Patients after Hormonal Treatment / Cultura de tecido ovariano para preservação da fertilidade em pacientes transexuais masculinos após tratamento hormonal

Abstract Objective To evaluate the reproductive and histological characteristics of fresh cultured ovarian tissue from transgender male patients. Methods An in vitro pilot study in which samples were collected during sex reassignment surgery for transgender male patients. The ovarian cortex was cut into fragments of 2 mm, 3mm, and 4 mm, and placed in a 96-well plate suitable for cultivation at days 0, 2, 4, 6, and 8, when the histology was analyzed. Results Stromal hyperplasia was observed in all samples, and it was not associated with the obtainment of primordial or primary follicles. Peripheral reduction in cell count was also a recurrent finding. Primordial and primary follicles were identified with a heterogeneous pattern in fragments from the same patient and from different patients, and follicles in more advanced stages of development (secondary and antral) were not found. There was an association between the diameter of the ovarian fragments and the identification of primary follicles (p=0.036). The number of days in culture was associated with histological signs of tissue damaging in the fragments (p=0.002). The total number of follicles identified in the samples with 2mm in diameter was significantly lower than in those that measured 4mm in diameter (p=0.031). Conclusion A diameter of 4mm is suitable for ovarian tissue culture with the benefit of ease of handling. Even after prolonged exposure to testosterone, the ovarian fragments presented primordial and primary follicles, maintaining viability throughout the days they were exposed to the culture. Freezing the ovarian cortex of transgender patients who will undergo surgery for gender reassignment would be an interesting option, in the future, for the preservation of fertility.

Resumo Objetivo Avaliar as características reprodutivas e histológicas de tecido ovariano cultivado a fresco de pacientes transexuais masculinos. Métodos Estudo experimental in vitro e piloto, no qual amostras foram coletadas durante a cirurgia de redesignação de sexo para pacientes transexuais masculinos. O córtex ovariano foi cortado em fragmentos de 2mm, 3mm, e 4mm, e colocado em placa de 96 poços própria para cultivo nos dias 0, 2, 4, 6 e 8, quando a histologia foi analisada. Resultados Hiperplasia estromal foi observada em todas as amostras, e não esteve associada à obtenção de folículos primordiais ou primários. A redução periférica no número de células também foi um achado recorrente. Folículos primordiais e primários foramidentificados com padrão heterogêneo emfragmentos domesmo paciente e em fragmentos de pacientes diferentes, não sendo encontrados folículos em estágios mais avançados de desenvolvimento (secundários e antrais). Houve associação entre o diâmetro dos fragmentos ovarianos e a identificação dos folículos primários (p=0,036). O número de dias de cultura esteve associado a sinais histológicos de lesão tecidual nos fragmentos (p=0,002). O número total de folículos identificados nas amostras de 2mm de diâmetro foi significativamente menor do que nas de 4mm de diâmetro (p=0,031). Conclusão O diâmetro de 4mm parece ser mais adequado para a cultura de tecido ovariano com a vantagem de fácil manejo. Mesmo após exposição prolongada à testosterona, os fragmentos ovarianos apresentavam folículos primordiais e primários, e manteve a viabilidade ao longo dos dias de exposição à cultura. No futuro, o congelamento da cortical do ovário de pacientes transgêneros que se submeterão à cirurgia de redesignação sexual poderia ser uma opção interessante para a preservação da fertilidade.

Report of a clinical and laboratory management of cell therapy for knee cartilage in the face of mycoplasma contamination

ABSTRACT To describe a case of autologous chondrocyte implantation after cell culture contamination by Mycoplasma pneumoniae and the measures taken to successfully complete cell therapy in a patient with focal chondral lesion. A 45-year-old male patient, complaining of chronic pain on the knee and no history of trauma. He had a chondral lesion in the trochlear region of the femur and clinical tests compatible with pain in the anterior compartment of the knee. Conservative treatment failed to alleviate symptoms. Surgical treatment was indicated, but due to the size of the lesion, membrane-assisted autologous chondrocyte implantation was the technique of choice. Cartilage biopsies were collected from the intercondylar region of the distal femur. After isolation, chondrocytes were expanded ex vivo in a trained laboratory, for three weeks, and seeded onto a commercially available collagen membrane prior to implantation in the knee. Two days before surgery, a cell culture sample tested positive for Mycoplasma pneumoniae. The source of contamination was found to be autologous blood serum, extracted from the patient´s peripheral vein, and used to supplement the cell culture medium. After treating the patient with antibiotics, all procedures were repeated and the new final cell product, free from contaminants, was successfully implanted. We discuss the strategies available to deal with this situation, and describe the results of this particular case, which led to modifications in the autologous chondrocyte implant protocol.

Actividad antimicrobiana de la peroxiadenosina en cultivos in vitro de Staphylococcus aureus resistente a meticilina / Antimicrobial activity of peroxyadenosine in vitro culture of resistant methicillin Staphylococcus aureus

RESUMEN Fundamento : las infecciones asociadas a la asistencia sanitaria y de la comunidad causadas por Staphylococcus aureus resistente a la meticilina, constituyen un problema de salud en el mundo. Esta situación se asocia al uso indiscriminado de antimicrobianos y a la mutagénesis bacteriana, por tanto, el desarrollo de nuevos productos con actividad antimicrobiana constituye una prioridad para Cuba. Objetivo : evaluar la actividad antimicrobiana de la peroxiadenosina en cultivo in vitro de Staphylococcus aureus resistente a la meticilina. Métodos : se realizó un estudio experimental de laboratorio en un modelo biológico (cultivo microbiano) en el Hospital Universitario Amalia Simoni de la provincia Camagüey, desde enero de 2016 hasta julio de 2020. La muestra se conformó con 60 cultivos biológicos de Staphylococcus aureus resistentes a la meticilina, mediante un muestreo aleatorio simple, distribuidos en dos grupos, cada uno con 30 cultivos. Un grupo correspondió a una cepa salvaje de Staphylococcus aureus resistentes a la meticilina, obtenida de la colección de cultivos del hospital y el otro a la cepa de referencia (ATCC 25923) de la American Type Culture Collection. Se estudiaron las variables: formación y concentración de la peroxiadenosina, preservación de la estructura aromática de la adenosina y el patrón de susceptibilidad a la meticilina. Resultados : la peroxiadenosina se formó a partir de la interacción del peróxido de hidrógeno con la adenosina. Los cultivos de Staphylococcus aureus resistentes a la meticilina se mostraron sensibles a las concentraciones de peroxiadenosina sin diluir y 1:2, demostrado por la presencia de halos de inhibición en el cultivo. Conclusiones : la inhibición del crecimiento de Staphylococcus aureus resistentes a la meticilina por la peroxiadenosina sugiere su evaluación como un nuevo producto antibacteriano eficaz contra este microorganismo.

ABSTRACT Background: infections associated with healthcare and the community caused by methicillin-resistant Staphylococcus aureus (MRSA) constitutes a health problem in the world. This situation is associated with the indiscriminate use of antimicrobials and bacterial mutagenesis, therefore, the development of new products with antimicrobial activity is a priority for Cuba. Objective: to evaluate the antimicrobial activity of peroxyadenosine in vitro culture of MRSA. Methods: an experimental laboratory study was carried out in a biological model (microbial culture) at the Amalia Simoni Hospital in Camagüey province, from January 2016 to July 2020. The sample consisted of 60 biological cultures through simple random sampling, distributed in two groups with 30 crops of each investigated layer. One group corresponded to a wild MRSA strain, obtained from the hospital culture collection and the other to the reference strain (ATCC 25923) from the American Type Culture Collection. The studied variables were: formation and concentration of peroxyadenosine, preservation of the aromatic structure of adenosine and the pattern of susceptibility to methicillin. Results: peroxyadenosine was formed from the interaction of hydrogen peroxide with adenosine. The MRSA cultures were sensitive to 1: 2 undiluted peroxyadenosine concentrations, demonstrated by the presence of inhibition halos in the culture. Conclusions: the inhibition of MRSA growth by peroxyadenosine suggests its evaluation as a new antibacterial product effective against this microorganism.

Preservation fluid cultures. Clinical significance in liver transplantation / Cultivos de líquido de preservación en trasplante hepático. Significancia clínica

Abstract The aim of this study was to determine the incidence of preservation fluids (PF) bacterial positive cultures, identify the germs involved, determine their correlation with infections in recipients during the postoperative period and compare outcomes in terms of morbidity, hospital stay and both patient and graft survival. We describe incidence and etiology of germs developed in PF cultures in our series and evaluate its impact on recipients. A prospective study in deceased donor liver transplants (LT) recipients was carried out from January 2014 to December 2017. Back table PF cultures were analized considering positive the development of any germs and negative to no signs of growth after 5 days. PF were classified as contamination or pathogens. Targeted antibiotic therapy was administered in the last ones. Recipients were divided in: PF (-) and PF(+). Recipients infections related to positive PF were analyzed. These were identified as "direct correlation" when the same germ grew up in PF. Hospital stay and 30 days follow up were compared. Eighty-eight patients PFs were included, 38% (33) had positive cultures, 28 (85%) of these were considered contamination and only 5 as pathogens. We found no differences in postoperative infections (p 0.840), ICU and total hospital stay (p 0.374 and 0.427) between both groups. Postoperative infections and hospital stay seem not to be influenced by PF cultures positivity. Treatment of isolated pathogens could have prevented infections, therefore, those groups that perform PF cultures should consider treatment in these cases and conclude prophylaxis when PF is negative or contaminated.

Resumen Las infecciones bacterianas son frecuentes en pacientes sometidos a trasplante hepático. Describimos la incidencia y etiología de los cultivos de líquidos de preservación (LP) positivos en nuestra serie y analizamos su importancia clínica. Se trata de un trabajo prospectivo de pacientes trasplantados hepáticos, entre enero 2014 a diciembre 2017. Se analizaron muestras de LP tomadas al finalizar la mesa de banco, considerándose positivo el desarrollo de cualquier germen y negativo la ausencia del mismo luego de 5 días. Los LP positivos se clasificaron en: con contaminantes y con patógenos. Los pacientes con LP patógenos recibieron tratamiento antibiótico de acuerdo al antibiograma. Los pacientes fueron divididos en dos grupos: con LP + y LP-. Las infecciones relacionadas a los LP fueron analizadas. Se consideró "correlación directa" cuando el mismo germen desarrolló en el LP y en el recipiente. Se comparó estadía hospitalaria en ambos grupos. Se incluyeron 88 pacientes, 38% (33) presentaron LP+, de los que el 85% (28) fueron por contaminación y 5 por pa tógenos. No se hallaron diferencias significativas en infecciones postoperatorias (p 0.840) y estadía hospitalaria (p 0.427) entre ellos. No hubo casos de "correlación directa". Las infecciones postoperatorias y la estadía hospitalaria de los pacientes no parecen estar influidas por la positividad de los cultivos de LP. El tratamiento dirigido a los gérmenes aislados como patógenos pudo prevenir infecciones, por lo tanto, los grupos que realizan cultivos de rutina deberían considerar el tratamiento en estos casos y finalizar la profilaxis cuando el LP sea negativo o contaminado.

Microbiological profile of leg ulcer infections: review study / Perfil microbiológico de las infecciones de ulceras de pierna: estudio de revisión / Perfil microbiológico das infecções de úlceras de perna: estudo de revisão

ABSTRACT Objectives: to analyze the microbiological profile of leg ulcers of patients treated at outpatient clinics and hospitals regarding the type of microorganism, microbiological selection of antibiotics, and techniques for the collection of culture material. Methods: literature review performed on LILACS, IBECS, MEDLINE, and CINAHL databases, resulting in a descriptive analysis of 27 studies. Results: 35.7% of the studies occurred in an outpatient care scenario; and 64.2% in hospitals. There was a predominance of swab (100%) in outpatient care and biopsy (55.5%) in the hospital. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were more common at both levels of assistance. Methicillin-resistant Staphylococcus aureus was isolated in both. Conclusions: the microbiological profile of infections was similar, with the presence of resistant bacteria in both environments. This fact causes concern and raises the need for research to elucidate it. The studies did not compare the effectiveness between biopsy and swab.

RESUMEN Objetivos: analizar perfil microbiológico de úlceras de pierna de pacientes atendidos en ambulatorio y hospital cuanto al tipo de microorganismo, selección microbiológica a los antibióticos y técnicas de recogida de material para cultura. Métodos: revisión de literatura realizada en bases LILACS, IBECS, MEDLINE y CINAHL, resultando en 27 estudios analizados descriptivamente. Resultados: ocurrieron en ambulatorio, 35,7% de los estudios; y en hospitales, 64,2%. Predominaron swab (100%) en ambulatorio y biopsia (55,5%) en hospital. Escherichia coli, Pseudomonas aeruginosa y Staphylococcus aureus fueron más comunes en dos niveles de asistencia. Hubo el aislamiento de Staphylococcus aureus resistente a la meticilina en ambos. Conclusiones: perfil microbiológico de las infecciones fue semejante, con presencia de bacterias resistentes en los dos ambientes. Ese hecho causa preocupación y suscita necesidad de investigaciones para elucidarlo. Estudios no compararon la efectividad entre biopsia y swab.

RESUMO Objetivos: analisar o perfil microbiológico de úlceras de perna de pacientes atendidos em ambulatório e hospital quanto ao tipo de microrganismo, seleção microbiológica aos antibióticos e técnicas de coleta de material para cultura. Métodos: revisão da literatura realizada nas bases LILACS, IBECS, MEDLINE e CINAHL, resultando em 27 estudos analisados descritivamente. Resultados: ocorreram em ambulatório, 35,7% dos estudos; e em hospitais, 64,2%. Predominaram swab (100%) em ambulatório e biópsia (55,5%) no hospital. Escherichia coli, Pseudomonas aeruginosa e o Staphylococcus aureus foram mais comuns nos dois níveis de assistência. Houve o isolamento de Staphylococcus aureus resistente à meticilina em ambos. Conclusões: o perfil microbiológico das infecções foi semelhante, com presença de bactérias resistentes nos dois ambientes. Esse fato causa preocupação e suscita necessidade de pesquisas para elucidá-lo. Os estudos não compararam a efetividade entre biópsia e swab.

Microbiological profile of leg ulcer infections: review study / Perfil microbiológico de las infecciones de ulceras de pierna: estudio de revisión / Perfil microbiológico das infecções de úlceras de perna: estudo de revisão

ABSTRACT Objectives: to analyze the microbiological profile of leg ulcers of patients treated at outpatient clinics and hospitals regarding the type of microorganism, microbiological selection of antibiotics, and techniques for the collection of culture material. Methods: literature review performed on LILACS, IBECS, MEDLINE, and CINAHL databases, resulting in a descriptive analysis of 27 studies. Results: 35.7% of the studies occurred in an outpatient care scenario; and 64.2% in hospitals. There was a predominance of swab (100%) in outpatient care and biopsy (55.5%) in the hospital. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were more common at both levels of assistance. Methicillin-resistant Staphylococcus aureus was isolated in both. Conclusions: the microbiological profile of infections was similar, with the presence of resistant bacteria in both environments. This fact causes concern and raises the need for research to elucidate it. The studies did not compare the effectiveness between biopsy and swab.

RESUMEN Objetivos: analizar perfil microbiológico de úlceras de pierna de pacientes atendidos en ambulatorio y hospital cuanto al tipo de microorganismo, selección microbiológica a los antibióticos y técnicas de recogida de material para cultura. Métodos: revisión de literatura realizada en bases LILACS, IBECS, MEDLINE y CINAHL, resultando en 27 estudios analizados descriptivamente. Resultados: ocurrieron en ambulatorio, 35,7% de los estudios; y en hospitales, 64,2%. Predominaron swab (100%) en ambulatorio y biopsia (55,5%) en hospital. Escherichia coli, Pseudomonas aeruginosa y Staphylococcus aureus fueron más comunes en dos niveles de asistencia. Hubo el aislamiento de Staphylococcus aureus resistente a la meticilina en ambos. Conclusiones: perfil microbiológico de las infecciones fue semejante, con presencia de bacterias resistentes en los dos ambientes. Ese hecho causa preocupación y suscita necesidad de investigaciones para elucidarlo. Estudios no compararon la efectividad entre biopsia y swab.

RESUMO Objetivos: analisar o perfil microbiológico de úlceras de perna de pacientes atendidos em ambulatório e hospital quanto ao tipo de microrganismo, seleção microbiológica aos antibióticos e técnicas de coleta de material para cultura. Métodos: revisão da literatura realizada nas bases LILACS, IBECS, MEDLINE e CINAHL, resultando em 27 estudos analisados descritivamente. Resultados: ocorreram em ambulatório, 35,7% dos estudos; e em hospitais, 64,2%. Predominaram swab (100%) em ambulatório e biópsia (55,5%) no hospital. Escherichia coli, Pseudomonas aeruginosa e o Staphylococcus aureus foram mais comuns nos dois níveis de assistência. Houve o isolamento de Staphylococcus aureus resistente à meticilina em ambos. Conclusões: o perfil microbiológico das infecções foi semelhante, com presença de bactérias resistentes nos dois ambientes. Esse fato causa preocupação e suscita necessidade de pesquisas para elucidá-lo. Os estudos não compararam a efetividade entre biópsia e swab.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Abstract Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Research progress in the 3D organoids of human pancreatic ductal adenocarcinoma / 国际生物医学工程杂志

Pancreatic ductal adenocarcinoma (PDA) organoids are 3D cultured from patient-derived stem cells or progenitor cells in vitro. PDA organoids have a variety of cell types, can realize structural self-organization through cell self-renewal, and are similar to the cells in the body of the original organ function in vivo biological bank. PDA organoids can be derived from surgical or biopsy tissue. The ability to build organoids from biopsy will facilitate the sampling of a larger population of PDA patients. Repeated sampling of patients can track the entire progression of the disease longitudinally. Compared with the traditional 2D cell culture and patient-derived xenotransplantation models, the three-dimensional culture of PDA organoids has the characteristics of short time and high success rate, and can be cryopreserved and maintain the stability of genetic traits. Organoids that can simulate diseases can be used as an alternative drug testing system. Using it for drug testing can not only better reflect the patient's response to drugs, but also can reduce the number of animal experiments. Moreover, when using organoids for testing, there is no need to understand the underlying molecular mechanism a priori, and chemical sensitivity testing can be performed directly, thereby shortening the testing time. In this paper, the advantages and disadvantages of different PDA organoids 3D culture methods and the verification methods for the stability and invasiveness of PDA organoids were reviewed. The mechanism of PDA organoids used for tumor chemotherapy drug sensitivity screening was discussed, and the application prospects and challenges of tumor biology in patient individualized treatment and precision medical treatment were discussed.

Detección de un mosaico de trisomía 21 en líquido amniótico / Detection of a mosaicism of trisomy 21 in amniotic liquid

Resumen Se analizó un resultado con alteración cromosómica tomado de una base de datos conformada por un total de 4755 muestras de líquido amniótico extraídos mediante amniocentesis con indicación de su médico tratante, riesgo sérico y edad materna avanzada. En este reporte se presenta la detección de un mosaico de trisomía 21 en líquido amniótico, mediante la técnica de Banda G donde se analizaron 20 metafases. Los resultados obtenidos documentan una composición cromosómica 47, XY+21 y 46, XY con una relación 9:11 respecto a las metafases analizadas, confirmándose así el diagnóstico del Síndrome de Down secundario a mosaico.

Abstract A result with chromosomal alteration was analyzed from a database consisting of a total of 4755 samples of amniotic fluid extracted by amniocentesis with indication of the attending physician, serum risk and advanced maternal age. This report presents the detection of a mosaicism of trisomy 21 in amniotic fluid, using G- Banding where 20 metaphases were analyzed. The results obtained document a chromosomal composition 47, XY + 21 and 46, XY with a 9:11 ratio with respect to the metaphases analyzed, confirming the diagnosis of Down syndrome secondary to mosaicism.

Avaliação dos efeitos do Bisfenol A em queratinócitos e células de neoplasia de cavidade oral / Evaluation of bisphenol A effects in keratinocytes and oral cavity neoplasia cells

O Bisfenol A (BPA) é um monômero utilizado na produção de garrafas plásticas, embalagens alimentícias, resinas odontológicas e vários outros materiais. Este monômero age como um desregulador do sistema endócrino e seus efeitos estão associados a cânceres em diferentes órgão e tecidos, como mama, próstata e tireóide. O BPA já foi detectado em fluidos humanos, incluindo saliva, mas os seus efeitos na mucosa bucal e em células orais neoplásicas não foram investigados. Os objetivos do presente trabalho foram 1) verificar os efeitos da exposição crônica ao BPA em glândulas salivares e mucosa bucal in vivo e 2) avaliar os efeitos do BPA in vitro em células de neoplasias bucais e queratinócitos; Para atender ao objetivo 1, camundongos machos e fêmeas receberam BPA (200 mg/mL) na água de beber durante 6 semanas. As mucosas orais (palato, língua e mucosa jugal) e as glândulas submandibulares foram avaliadas. Para atender ao objetivo 2, a resposta ao BPA foi examinada nas linhagens NOK-SI (queratinócito), HN12, HN13 (CCE de cavidade oral), UM HMC1 e UM HMC3a (neoplasia de glândula salivar). Os seguintes parâmetros foram avaliados: viabilidade, proliferação, invasão, angiogênese, produção de citocinas e fatores de crescimento e possíveis mecanismos de ação do BPA. Resultados. A exposição de camundongos ao BPA resultou em alterações microscópicas caracterizadas pelo aumento da espessura do epitélio da mucosa oral (palato, língua e mucosa jugal) e uma redução no número de ácinos das glândulas submandibulares. Foi observado também um acúmulo de BPA nos tecidos orais. In vitro, nas linhagens de CCE, o BPA aumentou a proliferação, invasão celular e os níveis da proteína vimentina, e ainda induziu a secreção de citocinas e fatores de crescimento, e a acetilação de histonas H3. Em queratinócitos orais, o BPA aumentou a proliferação celular e induziu a secreção de fatores de crescimento e a expressão de receptores de estrógeno (ER) α e ß. Os efeitos do BPA foram revertidos na presença do antagonista puro do ER. Nas linhagens de neoplasias de glândula o BPA não alterou a proliferação e induziu a expressão de p63. Em conclusão, o BPA induz alterações morfológicas nos tecidos bucais e alterações moleculares nos queratinócitos e nas células de CCE de cavidade oral. Os mecanismos pelos quais o BPA induz estas alterações são dependentes da interação BPA-ER e da acetilação de histonas.

Bisphenol A (BPA) is a monomer used to product plastic bottles, food packaging, inner coating of food cans, thermal papers, medical devices, dental resins and various other materials. Due to its chemical structure, this monomer acts as a deregulator of the endocrine system and its effects are associated with cancers in different organs and tissues such as breast, endometrium, ovary, prostate, testis and thyroid. BPA has been detected in several human fluids, including saliva, however its effects on the normal oral mucosa and neoplastic oral cells have not been investigated yet. Thus, the objectives of the present study were 1) to verify the effects of chronic exposure to BPA in salivary glands and oral mucosa in vivo and 2) to evaluate the effects of BPA in vitro on oral tumor cells and keratinocytes. To meet objective 1, male and female mice received BPA (200 mg / mL) in drinking water for 6 weeks. The oral mucosa (palate, tongue and buccal mucosa) and submandibular salivary glands were evaluated microscopically. To meet objective 2, the response to BPA was examined in immortalized cell lines NOK SI (keratinocyte); HN12, HN13 (OSCC), UM HMC1 and UM HMC3a (salivary gland tumor). The following parameters were evaluated: viability, proliferation, invasion, angiogenesis, cytokine and growth factors production. Results. Exposure of mice to BPA resulted in microscopic changes characterized by increased thickness of the oral mucosa epithelium (palate, tongue and buccal mucosa) and a reduction in the number of submandibular salivary glands acini. There was also an accumulation of BPA in the oral tissues. In vitro, in OSCC cells, BPA increased cell proliferation and invasion, vimentin expression, induced secretion of cytokines and growth factors, and induced histone H3 acetylation. In oral keratinocytes, BPA increased cell proliferation and induced secretion of growth factors and estrogen receptor (ER) α and ß expression. The effects of BPA were reversed in the presence of the pure ER antagonist. In salivary gland tumor cell lines, BPA did not alter the proliferation and induced the expression of p63. BPA mechanism of action involves its interaction with ER, since the effects were reverted in the presence of pure receptor antagonist. In conclusion, BPA induces morphological changes in oral tissues and molecular changes in keratinocytes and OSCC cells. The mechanisms which BPA induces these changes are dependent to the BPA-ER interaction and histone acetylation.

Blefarite: epidemiologia, etiologia, apresentações clínicas, tratamento e evolução de nossos pacientes / Blepharitis: epidemiology, etiology, clinical presentations, treatment and evolution of our patients

Resumo Objetivo: A blefarite é uma das condições mais comumente encontradas na prática oftalmológica e se constitui em uma causa frequente de irritação e desconforto ocular. Por ser uma doença de difícil tratamento, os autores buscaram compreender melhor a epidemiologia, etiologia, apresentações clínicas, tratamento e evolução de seus pacientes, visando maior sucesso terapêutico. Métodos: Foram avaliados retrospectivamente e transversalmente o prontuário de 124 pacientes do Centro de Oftalmologia Tadeu Cvintal, os quais apresentavam blefarite e foram submetidos à classificação de gravidade e coleta de secreções palpebrais para cultura bacteriana e antibiograma. Resultados: A media da idade dos pacientes foi de 67,4 anos, o sexo feminino foi responsável por 70 (56,4%) casos e o masculino por 54 (43,5%). Quanto à gravidade da doença, constatou-se 71 casos de blefarite leve (56,8%), 52 (41,6%) com intensidade moderada e 2 (1,6%) casos graves. Avaliando o seguimento do tratamento da doença, foi observado que 103 (82,4%) pacientes não retornaram para avaliar o resultado do tratamento e apenas 22 (17,6%) retornaram. Em relação às culturas realizadas, 82 (66,1%) não apresentaram crescimento microbiano. Dentre as 42 (33,8%) amostras positivas, os Staphylococcus coagulase negativo foram os mais prevalentes, sobretudo os Staphylococcus epidermidis, responsável por 35 (83,3%) delas. Quanto à sensibilidade aos antibióticos, os agentes de nossa amostra demonstraram maior resistência à Penicilina, Eritromicina e Ciprofloxacino e 100% de sensibilidade à Linezolida, Vancomicina e Daptomicina. Conclusão: Conhecendo melhor as características epidemiológicas da blefarite e a sensibilidade antimicrobiana das bactérias envolvidas, é possível oferecer tratamentos mais eficazes.

Abstract Objective: Blepharitis is one of the most commonly encountered conditions in ophthalmic practice and is a frequent cause of eye irritation and discomfort. Being a difficult to treat disease, the authors sought to better understand the epidemiology, etiology, clinical presentations, treatment and evolution of their patients, aiming at greater therapeutic success. Methods: The medical records of 124 patients of Centro de Oftalmologia Tadeu Cvintal who had blepharitis were retrospectively and cross-sectionally evaluated and underwent severity classification and collection of eyelid secretions for bacterial culture and antibiogram. Results: The mean age of the patients was 67.4 years, females accounted for 70 (56.4%) cases and males for 54 (43.5%). Regarding the severity of the disease, there were 71 cases of mild blepharitis (56.8%), 52 (41.6%) with moderate intensity and 2 (1.6%) severe cases. Evaluating the follow-up of treatment of the disease, it was observed that 103 (82.4%) patients did not return to evaluate the treatment outcome and only 22 (17.6%) returned. In respect of the cultures performed, 82 (66.1%) did not show microbial growth. Among the 42 (33.8%) positive samples, coagulase-negative staphylococci were the most prevalent, especially Staphylococcus epidermidis, responsible for 35 (83.3%) of them. As for antibiotic sensitivity, the agents in our sample showed greater resistance to Penicillin, Erythromycin and Ciprofloxacin and 100% sensitivity to Linezolid, Vancomycin and Daptomycin. Conclusion: By better understanding the epidemiological characteristics of blepharitis and the antimicrobial sensitivity of the bacteria involved, it is possible to offer more effective treatments.

Cytotoxicity and biocompatibility of a new bioceramic endodontic sealer containing calcium hydroxide

Abstract: This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.

Isolation and characterization of a human cementocyte-like cell line, HCY-23

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

Subgingival microbiological profile of periodontitis patients in Dominican Republic

The aim of this study was to evaluate the deproteinization of primary enamel by analyzing etching pattern types, with and without the application of 5% NaOCl before acid etching with 37% H3PO4. Fifteen extracted human primary molars were randomly selected for the present in vitro study; 1mm x 1mm blocks were prepared and divided into two groups (n = 21). These groups were treated as follows: Group AAcid Etching with 37% H3PO4 gel for 15 s; Group B5% NaOCl for 60 s + Acid Etching with 37% H3PO4for 15 s. The specimens were prepared for scanning electron microscopy analysis. The images were evaluated for quality types I and II etching of the enamel surface using ImageJ software. Datasets were checked for normality by KolgomorvSmirnov test and the nonparametric unpaired MannWhitney test was applied. The mean surface area of type I and II etching pattern values was 1922.314 µm2for Group A and 3840.473 µm2Group B. We conclude that deproteinization with 5% NaOCl prior to acid etching can be used to increase the area of adhesion and the quality of the etching pattern (AU)

El objetivo del estudio fue evaluar la desproteinización del esmalte primario a través de los tipos de patrones de grabado, con y sin NaOCl 5% utilizado antes del grabado ácido con H3PO4 37%. Quince dientes primarios humanos extraídos se seleccionaron al azar para el presente estudio in vitro, se prepararon bloques de 1mm x 1 mm y se dividieron en dos grupos (n = 21). Estos grupos se trataron de la siguiente manera: Grupo A: Grabado ácido con H3PO4 37% en gel durante 15 segundos; Grupo B: NaOCl 5% durante 60 segundos + Grabado ácido con H3PO4 37% durante 15 segundos. Las muestras se prepararon para el análisis de microscopía electrónica de barrido. Las imágenes obtenidas se evaluaron principalmente por la calidad de los grabados tipo I y II de la superficie del esmalte primario, utilizando el software Image J. Los datos se analizaron en cuanto a su normalidad mediante la prueba de KolgomorvSmirnov, se utilizó pruebas no paramétricas: Prueba de MannWhitney no pareada. Como resultado, se encontró que el área de superficie media de los valores de patrón de grabado de tipo I y II para el Grupo A era 1922,314 µm2 y el Grupo B era 3840,473 µm2. Finalmente, llegamos a la conclusión de que se puede usar la desproteinización con NaOCl 5% antes del grabado ácido para aumentar el área de adhesión y la calidad del patrón de grabado (AU)

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.