Medicinal plant extracts interfere in gastric cancer stem cells fluorescence-based assays.

Fluorescence is used in various biological assays due to its high sensitivity, versatility, and precision. In recent years, studies using medicinal plant extracts have increased. However, fluorescence-based assays could be biased by plant metabolites autofluorescence. To address this issue, this study investigated the interference caused by methanolic extracts and chloroform fractions of three medicinal plants in three fluorescence-based assays on gastric cancer stem cells(CSC): resazurin reduction, confocal microscopy, and flow cytometry. CSC were isolated based on CD44 surface marker, incubated with methanolic extracts and chloroform fractions of Buddleja incana, Dracontium spruceanum, Piper aduncum. Resazurin assay evidenced that CSC exposed to extracts and fractions from the three plants showed significant differences in relative fluorescence units (RFU) levels (p < 0.001) compared to the unexposed groups after a 3-hour incubation. In addition, DMSO-treated CSC exposed to extracts and fractions had significantly lower fluorescence levels than living ones, but higher than extracts and fractions without cells. In confocal microscopy, cancer stem cells exposed to extracts and fractions of B. incana and P. aduncum were observed in the same emission spectra of the CSC markers. In flow cytometry, CSC exposed to extracts and fractions without any fluorescent dyes were detected in the double positive quadrants for CSC markers (CD44+/CD133 + ). Among the three plants, D. spruceanum exhibited the least interference. These results show that methanolic extracts and chloroform fractions contain autofluorescent metabolites that interfere with fluorescence-based assays. These results highlight the importance of a prior evaluation for possible fluorescence interference to avoid interpretation biases in fluorescence assays.

Cytokine Profiles Associated With Worse Prognosis in a Hospitalized Peruvian COVID-19 Cohort.

Cytokines, chemokines and growth factors present different expression profiles related to the prognosis of COVID-19. We analyzed clinical parameters and assessed the expression of these biomarkers in patients with different disease severity in a hospitalized Peruvian cohort to determine those associated with worse prognosis. We measured anti-spike IgG antibodies by ELISA and 30 cytokines by quantitative suspension array technology in 123 sera samples. We analyzed differences between patients with moderate, severe and fatal COVID-19 by logistic regression at baseline and in longitudinal samples. Significant differences were found among the clinical parameters: hemoglobin, neutrophils, lymphocytes and C-reactive protein (CRP), creatinine and D-dimer levels. Higher anti-spike IgG antibody concentrations were associated to fatal patient outcomes. At hospitalization, IL-10, IL-6, MIP-1α, GM-CSF, MCP-1, IL-15, IL-5, IL1RA, TNFα and IL-8 levels were already increased in fatal patients´ group. Meanwhile, multivariable analysis revealed that increased GM-CSF, MCP-1, IL-15, and IL-8 values were associated with fatal outcomes. Moreover, longitudinal analysis identified IL-6 and MCP-1 as the main risk factors related to mortality in hospitalized COVID-19 patients. In this Peruvian cohort we identified and validated biomarkers related to COVID-19 outcomes. Further studies are needed to identify novel criteria for stratification of SARS-CoV-2 infected patients at hospital entry. Background: In the most severe forms of SARS-CoV-2 infection, large numbers of innate and adaptive immune cells become activated and begin to produce pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation. Methods: A total of 55 patients with laboratory-confirmed COVID-19 admitted to the Hospital Nacional Guillermo Almenara Irigoyen in Lima, Peru were enrolled during August-October 2020. Of these, 21 had moderate disease, 24 severe diseases and 10 died. We measured 30 cytokines and chemokines by quantitative suspension array technology and anti-spike IgG antibodies using a commercial ELISA. We evaluated these parameters in peripheral blood every 2-5 days until patient discharge or death. Patient information and clinical parameters related were obtained from the respective clinical histories. Results: The frequency of obesity differed among the 3 groups, being most frequent in patients who died. There were also significant differences in clinical parameters: hemoglobin, segmented neutrophils, lymphocytes,C-reactive protein, creatinine and D-dimer levels. Greater anti-spike IgG antibody concentrations were associated to fatal outcomes. In univariate analyses, higher baseline concentrations of IL-6, MIP-1α, GM-CSF, MCP-1, IL-15, IL-5, IL1RA, TNFα, IL-8 and IL-12p70 correlated with severity, while multivariable analysis showed that increased concentrations in 4 biomarkers (GM-CSF, MCP-1, IL-15, IL-8) were associated with fatal outcomes. Longitudinal analysis showed IL-6 (hazard ratio [HR] 6.81, 95% confidence interval [CI] 1.6-28.7) and MCP-1 (HR 4.61, 95%CI 1.1-19.1) to be related to mortality in hospitalized COVID-19 patients. Conclusions: Cytokine, chemokine and growth factor profiles were identified and validated related to severity and outcomes of COVID-19. Our findings may be useful to identify novel criteria for COVID-19 patient stratification at hospital entry.

Cytotoxic activity of the chloroform fraction of Piper aduncum and its effect on the cell cycle in gastric cancer cell lines. / Actividad citotóxica de la fracción clorofórmica de Piper aduncum y su efecto en el ciclo celular en líneas celulares de cáncer gástrico.

OBJECTIVES: To evaluate the cytotoxic activity of the chloroform fraction of the Piper aduncum methanolic extract (PAMoCl) and its effect on the cell cycle in two gastric cancer cell lines: AGS and KATO III. MATERIALS AND METHODS: The cytotoxic effect of PAMoCl was evaluated in cell lines AGS and KATO III. The following PAMoCl concentrations were tested, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL. Resazurine was used to evaluate cell viability. In the cell cycle assay, the cells were treated with 19.62 µg/mL and 39.23 µg/mL of PAMoCl for AGS as well as 87.49 µg/mL and 160 µg/mL for KATO III. After 24 hours both cell lines were analyzed by flow cytometry. RESULTS: PAMoCl showed cytotoxic activity, inhibiting cell growth by 50%. It presented a (IC50) of 39.23 µg/mL and 87.49 µg/mL at 24 hours and a (IC50) of 49.47 µg/mL and 64.68 µg/mL at 48 hours against AGS and KATO III cell lines, respectively. In addition, it was observed that PAMoCl has an effect on the cell cycle, it causes an accumulation of cells in the G2/M phase. CONCLUSIONS: PAMoCl contains secondary metabolites with cytotoxic activity that have an effect on the G2/M phase of the cell cycle, in two gastric cancer cell lines, both primary and metastatic. The results of this study will allow us to deepen the search for more effective active ingredients found in PAMoCl for eliminating gastric cancer cells, but with less toxicity for healthy cells.

OBJETIVOS: Evaluar la actividad citotóxica de la fracción clorofórmica del extracto metanólico de Piper aduncum (PAMoCl) y su efecto en el ciclo celular en dos líneas celulares de cáncer gástrico: AGS y KATO III. MATERIALES Y MÉTODOS: El efecto citotóxico de PAMoCl se evaluó en las líneas celulares: AGS y KATO III. Se probaron concentraciones de PAMoCl: 1,25; 2,5; 5; 10; 20; 40; 80 y 160 µg/mL. Para evaluar la viabilidad celular se usó el reactivo resazurina. En el ensayo de ciclo celular las células fueron tratadas con 19,62 µg/mL y 39,23 µg/mL de PAMoCl para AGS, así como 87,49 µg/mL y 160 µg/mL para KATO III. Después de 24 horas ambas líneas celulares fueron analizadas por citometría de flujo. RESULTADOS: PAMoCl mostró actividad citotóxica con una inhibición del crecimiento celular en un 50% (IC50) de 39,23 µg/mL y 87,49 µg/mL a las 24 horas y un IC50 de 49,47 µg/mL y 64,68 µg/mL a las 48 horas frente a las líneas celulares AGS y KATO III, respectivamente. Además, se observó que PAMoCl tiene efecto a nivel del ciclo celular: provoca una acumulación de células en la fase G2/M. CONCLUSIONES: PAMoCl contiene metabolitos secundarios con actividad citotóxica que tienen efecto en la fase G2/M del ciclo celular, en dos líneas celulares de cáncer gástrico tanto primario como metastásico. Los resultados de este estudio permitirán profundizar en la búsqueda de principios activos presentes en PAMoCl que tengan mayor eficacia en la eliminación de células de cáncer gástrico, pero con menor toxicidad en células sanas.

[Procedure for culture and identification of stem cells from human lipoaspirate for research purposes]. / Procedimiento para el cultivo e identificación de células madre obtenidas de lipoaspirado humano con fines de investigación.

Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.

Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.

Actividad citotóxica de la fracción clorofórmica de Piper aduncum y su efecto en el ciclo celular en líneas celulares de cáncer gástrico / Cytotoxic activity of the chloroform fraction of Piper aduncum and its effect on the cell cycle in gastric cancer cell lines

RESUMEN Objetivos: Evaluar la actividad citotóxica de la fracción clorofórmica del extracto metanólico de Piper aduncum (PAMoCl) y su efecto en el ciclo celular en dos líneas celulares de cáncer gástrico: AGS y KATO III. Materiales y métodos: El efecto citotóxico de PAMoCl se evaluó en las líneas celulares: AGS y KATO III. Se probaron concentraciones de PAMoCl: 1,25; 2,5; 5; 10; 20; 40; 80 y 160 µg/mL. Para evaluar la viabilidad celular se usó el reactivo resazurina. En el ensayo de ciclo celular las células fueron tratadas con 19,62 µg/mL y 39,23 µg/mL de PAMoCl para AGS, así como 87,49 µg/mL y 160 µg/mL para KATO III. Después de 24 horas ambas líneas celulares fueron analizadas por citometría de flujo. Resultados: PAMoCl mostró actividad citotóxica con una inhibición del crecimiento celular en un 50% (IC50) de 39,23 µg/mL y 87,49 µg/mL a las 24 horas y un IC50 de 49,47 µg/mL y 64,68 µg/mL a las 48 horas frente a las líneas celulares AGS y KATO III, respectivamente. Además, se observó que PAMoCl tiene efecto a nivel del ciclo celular: provoca una acumulación de células en la fase G2/M. Conclusiones: PAMoCl contiene metabolitos secundarios con actividad citotóxica que tienen efecto en la fase G2/M del ciclo celular, en dos líneas celulares de cáncer gástrico tanto primario como metastásico. Los resultados de este estudio permitirán profundizar en la búsqueda de principios activos presentes en PAMoCl que tengan mayor eficacia en la eliminación de células de cáncer gástrico, pero con menor toxicidad en células sanas.

ABSTRACT Objectives: To evaluate the cytotoxic activity of the chloroform fraction of the Piper aduncum methanolic extract (PAMoCl) and its effect on the cell cycle in two gastric cancer cell lines: AGS and KATO III. Materials and methods: The cytotoxic effect of PAMoCl was evaluated in cell lines AGS and KATO III. The following PAMoCl concentrations were tested, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg/mL. Resazurine was used to evaluate cell viability. In the cell cycle assay, the cells were treated with 19.62 μg/mL and 39.23 μg/mL of PAMoCl for AGS as well as 87.49 μg/mL and 160 μg/mL for KATO III. After 24 hours both cell lines were analyzed by flow cytometry. Results: PAMoCl showed cytotoxic activity, inhibiting cell growth by 50%. It presented a (IC50) of 39.23 μg/mL and 87.49 μg/mL at 24 hours and a (IC50) of 49.47 μg/mL and 64.68 μg/mL at 48 hours against AGS and KATO III cell lines, respectively. In addition, it was observed that PAMoCl has an effect on the cell cycle, it causes an accumulation of cells in the G2/M phase. Conclusions: PAMoCl contains secondary metabolites with cytotoxic activity that have an effect on the G2/M phase of the cell cycle, in two gastric cancer cell lines, both primary and metastatic. The results of this study will allow us to deepen the search for more effective active ingredients found in PAMoCl for eliminating gastric cancer cells, but with less toxicity for healthy cells.

Procedimiento para el cultivo e identificación de células madre obtenidas de lipoaspirado humano con fines de investigación / Procedure for culture and identification of stem cells from human lipoaspirate for research purposes

RESUMEN Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.

ABSTRACT Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.