Tumor antigen-loaded dendritic cells combined with cytokine-induced killer (CIK) enhance the killing activity of human esophageal cancer cells by promoting ASK1 activation / 细胞与分子免疫学杂志

Objective To clarify the effect and mechanism of tumor antigen-loaded dendritic cells (Ag-DCs) combined with cytokine-induced killers (CIKs) on the killing of esophageal cancer tumor cells. Methods Peripheral blood DCs and CIKs were induced and cultured, and the DCs were loaded with tumor antigen to obtain Ag-DCs, and Ag-DCs were co-cultured with CIKs. The experiment was divided into CIK group, DC combined with CIK group, Ag-DC combined with CIK group. Flow cytometry was used to detect the phenotype of cells. MTT assay was employed to determine the killing activity against EC9706 cells. Annexin V-FITC/PI double staining was used to detect the apoptosis rate of cells, immunofluorescence staining to detect the expression of phosphorylated apoptotic signal-regulated kinase 1 (p-ASK1) and Western blot analysis to detect the expression of ASK1 pathway related proteins. A nude mouse model of esophageal cancer transplantation tumor was constructed and divided into control group, DC combined with CIK group and Ag-DC combined with CIK group. The corresponding immune cells were injected into the tail vein for treatment and the tumor volume was measured every 2 days. After 21 days, all nude mice were sacrificed with the tumors taken out. HE staining was used to observe the tumor pathological changes and immunohistochemical staining was performed to detect the expression of ki67 and ASK1 in the tumor tissue. Results Comparedwith the CIK group alone and the DC combined with CIK group, the ratio of CD3+ CD8+ and CD3+ CD56+ in the cells significantly increased after Ag-DCs and CIKs co-culture, along with the increased killing rate of EC9706 cells, increased apoptosis rate of EC9706 cells, and the improved activation level of ASK1. Compared with the CIK group and the DC combined with CIK group, the growth of the transplanted tumor in nude mice treated with Ag-DCs combined with CIKs was significantly inhibited, and after 21 days, it was observed that the tumor tissue mass in this group was relatively smaller, with sparsely arranged cells in the tumor tissue and a decline in the positive rate of ki67 in tumor tissue, while the positive rate of ASK1 was significantly increased. Conclusion Co-cultivation of tumor antigen-loaded DCs with CIKs can significantly increase the killing activity of esophageal cancer tumor cells. The mechanism of action may be related to the activation of the ASK1 pathway.

Cytotoxic effect of hydroalcoholic extract of Annona Muricata against a human cell line of gastric adenocarcinoma / Efecto citotóxico del extracto hidroalcoholico de la Annona Muricata frente a la línea celular humana de adenocarcinoma gástrico

Background: Therapeutic advances against cancer have not been as successful as expected and have adverse effects that patients rarely tolerate. A study in Peru identified favorable anticancer effects of Annona muricata (AM), a medicinal plant known as soursop, in C-678 mouse gastric adenocarcinoma. However, to date, no results have been reported in human cells. Objective: The objective of this study was to determine the cytotoxic effect of AM extract against a human gastric adenocarcinoma cell line (AGS). Methodology: Experimental in vitro analytical study using a hydroalcoholic extract of AM (AMOH) leaves collected in the Amazonas. Chemical functional groups were identified by phytochemical screening. To obtain the cytotoxic effect, different dilutions of extract were added to the plates containing the cell lines and the data were extrapolated to GraphPad employing an observation card. Finally, the cytotoxic effect was expressed as the half-maximal inhibitory concentration (IC50) and nonlinear regression analysis was performed to determine the growth inhibition of cancer cells. Results: Phytochemical screening showed the presence of reducing carbohydrates, alkaloids, phenols, tannins, triterpenes, steroids, saponins, flavonoids, proteins, cardiac glycosides, and anthocyanins. A calibration curve with gallic acid was used to determine the total phenol content and, quercetin was used to identify the flavonoid content. The AGS cell line showed cytotoxic activity with AMOH with an IC50 at 24 hours of 45.81 µg/mL and 19.05 µg/mL at 48 hours. Conclusion: Several chemical functional groups of AM were identified. In addition, the AMOH showed a cytotoxic effect against the AGS cell line

Antecedente: Los avances terapéuticos frente al cáncer no han tenido el éxito esperado y presentan efectos adversos pocas veces tolerados por el paciente. Un estudio en Perú identificó el efecto anticancerígeno de la Annona muricata (AM), planta medicinal conocida como guanábana, en adenocarcinoma gástrico de ratón C-678 con resultados favorables, sin embargo, no se ha encontrado evidencia previa en células humanas. Objetivo: El objetivo de este estudio fue determinar el efecto citotóxico del extracto de AM frente a la línea celular de adenocarcinoma gástrico humano (AGS). Materiales y métodos: Estudio experimental in vitro tipo analítico con extracto hidroalcohólico de hojas de AM (AMOH) recolectadas en Amazonas. Mediante screening fitoquímico se identificaron los grupos funcionales químicos. Para obtener el efecto citotóxico, se añadieron diferentes diluciones de extracto a las placas que contienen las líneas celulares y mediante una ficha de observación los datos fueron extrapolados a GraphPad. Finalmente se expresó como la concentración inhibitoria media máxima (IC50) y se hizo un análisis de regresión no lineal con la finalidad de encontrar la cantidad de inhibición de crecimiento de células oncológicas. Resultados: En el screening fitoquímico se pudo identificar la presencia de carbohidratos reductores, alcaloides, fenoles, taninos, tritérpenos y esteroides, saponinas, flavonoides, proteínas, glicósidos cardiotónicos y antocianinas. Para identificar el contenido total de fenoles se utilizó la curva de calibración con ácido gálico el cual nos comprobó la presencia de una buena cantidad de estos metabolitos. Adicionalmente se utilizó quercetina para identificar el contenido de flavonoides, obteniendo resultados favorables ya que se hizo evidente su presencia. La línea celular AGS mostró una actividad citotóxica frente al AMOH con un IC50 a las 24 horas de 45.81ug/mL y 19.05ug/mL a las 48 horas. Conclusión: Se identifica a los grupos funcionales de la AM. Además, AMOH demostró un efecto citotóxico contra la línea celular AGS

Assessment of cytotoxicity, genotoxicity, and mutagenicity of the dexchlorpheniramine reference standard and pharmaceutical formula in human peripheral blood mononuclear cells

Abstract Dexchlorpheniramine is a first-generation classical antihistamine, clinically used to treat allergies. The main objective of our study was to evaluate the effects of the dexchlorpheniramine reference standard (DCPA Ref. St) and a pharmaceutical formula on DNA in human peripheral blood mononuclear cells (PBMCs). We exposed PBMCs to five different concentrations (0.5, 2.5, 5, 10, and 50 ng/mL) of DCPA Ref. St DCPA Ref. St and pharmaceutical formula in order to evaluate their cytotoxic, genotoxic, and mutagenic potential. The results showed that both dexchlorpheniramine formulations did not affect PBMC viability and CD3+, CD4+, or CD8+ lymphocyte subpopulations. The DCPA Ref. St and pharmaceutical formula neither induced genotoxic or mutagenic effects nor numerical or structural chromosomal alterations in PBMCs after 24 hours of exposure.

Effects of hydroxyurea on cytotoxicity, inflammation and oxidative stress markers in neutrophils of patients with sickle cell anemia: dose-effect relationship

ABSTRACT Introduction: Although the efficacy of hydroxyurea (HU) in inhibiting erythrocyte sickling has been well demonstrated, the action of this drug on human neutrophils and the mechanism by which it improves the manifestations of the disease have not been studied thoroughly. We aimed to investigate the cell viability, along with inflammatory and oxidative markers in the neutrophils of sickle cell anemia (SCA) patients and the effects of HU therapy on these cells, by evaluating the dose-responsiveness. Methods: In the present study, 101 patients (45 men and 56 women, aged 18-69 years) with SCA were divided into groups according to the use or not of HU: the SS group (without HU treatment, n = 47) and the SSHU group (under HU treatment, n = 54). The SSHU group was further stratified into subgroups according to the daily dose of the drug that patients already used: SSHU - 0.5 g (n = 19); SSHU - 1 g (n = 26) and SSHU - 1.5-2 g (n = 9). A control group (AA) comprised 50 healthy individuals. Neutrophils isolated from whole blood were analyzed using Trypan Blue, monoiodotyrosine (MTT) and lactate dehydrogenase (LDH) toxicity assays. Myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and concentrations of interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) and malonaldehyde (MDA) were also measured. Results: Neutrophils from SCA patients showed membrane fragility and a significant decrease in cell viability when analyzed by Trypan Blue (p < 0.05), MTT (p < 0.001) and LDH (p = 0.011), compared to the AA group. Levels of inflammatory (MPO, TNF-α, and IL-10) and oxidative markers (SOD, GSH-Px, and MDA) were also altered (p < 0.05) in these cells, showing a significant difference in the SSHU-1g and SSHU - 1.5-2 g groups, compared to the SS group. Treatment with HU reverted the levels of all markers to concentrations similar to those in healthy individuals in a positive dose-effect relationship. Conclusion: The HU did not generate a cytotoxic effect on neutrophils in SCA patients, but it modulated their oxidative and inflammatory mechanisms, promoting cytoprotection with a positive dose-effect.

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Citotoxicidade, genotoxicidade e expressão gênica em células-tronco mesenquimais expostas a materiais endodônticos / Cytotoxicity, genotoxicity and gene expression in mesenchymal stem cells exposed to endodontic cements

No mercado encontramos uma grande variedade de materiais endodônticos disponibilizados para uso clínico, mas diversos estudos mostram divergências de opiniões com relação ao comportamento biológico dos diferentes materiais. Este trabalho teve como objetivos investigar a viabilidade celular, a expressão de genes envolvidos na plasticidade celular e a diferenciação celular em culturas de células- tronco recuperadas de polpa dentária humana (hDPSCs) quando em contato com quatro materiais endodônticos (Endofill, Pulp Canal Sealer, Sealer 26, MTA) rotineiramente utilizados na clínica odontológica. Objetivou também, por meio de uma revisão sistemática, analisar a biocompatibilidade de cimentos de uso endodôntico sobre células tronco de origem dental. Para isto, o metabolismo celular das hDPSCs, quando em contato com os capilares contendo ou não os cimentos, foi avaliado pelo ensaio de MTT (24 e 48 horas) e a viabilidade celular foi analisada pelo ensaio de exclusão do azul de tripan (48 horas). A plasticidade celular, na presença dos capilares contendo ou não os cimentos, foi avaliada pela expressão gênica dos marcadores CD34, CD45, Nestin, CD105, Nanog e OCT-4 por PCR. Finalmente, a diferenciação celular frente aos cimentos endodônticos foi verificada pela expressão dos genes RUNX2, ALP, OC/BGLAP e DMP1 por RT-PCR. Os dados foram analisados pelo teste ANOVA com correção de Bonferroni (p<0.05). Observou-se que os cimentos Pulp Canal Sealer e o Endofill reduziram significativamente a viabilidade e o metabolismo celular quando comparados ao controle após 48 horas (p<0.001). O MTA e o Sealer 26 não interferiram na viabilidade celular em ambos os períodos de avaliação (p>0.05). As hDPSCs, quando cultivadas na presença do MTA e Sealer 26, expressaram os marcadores Nestin, CD105, NANOG e OCT-4, e não expressaram CD34 e CD45. Por sua vez, o MTA e o Sealer 26 interferiram positivamente ou negativamente na expressão gênica de DMP1, OC/BGLAP e RUNX2 em relação ao grupo controle (p<0.05), mas não houve diferença significativa em relação à expressão gênica de ALP (p>0.05). Portanto, MTA e Sealer 26 demonstram boa compatibilidade biológica quando na presença das hDPSCs. A revisão sistemática demonstrou que a maioria dos materiais, apresentam boa compatibilidade quando em contato com as células tronco, estando aptos a serem utilizados na prática clínica.

On the market, we found a wide variety of endodontics cements available for clinical use, but several studies show divergences of opinion regarding the biological behavior of these different materials. This work aimed to investigate cell viability and metabolism, an expression of genes involved in cell plasticity and cell differentiation in stem cell cultures recovered from human dental pulp (hDPSCs) when in contact with four endodontic cements (Endofill, MTA, Pulp Canal Sealer, Sealer 26) routinely used in endodontic clinic. It also aimed, through a systematic review, to analyze the biocompatibility of endodontic materials on dental stem cells. For this, the viability and metabolism of hDPSCs, when it comes into contact with capillaries that included or not cements, was assessed by MTT assay (24 and 48 hours) and exclusion of trypan blue assay (48 hours). Cellular plasticity, with the presence of capillaries containing or not sealers, was evaluated by the genetic expression of the markers CD34, CD45, Nestin, CD105, Nanog and OCT-4 by PCR. Finally, cell differentiation from endodontics sealers was verified by the expression of the RUNX2, ALP, OC/BGLAP and DMP1 genes by RT-PCR. The data were analyzed using the ANOVA test with Bonferroni correction (p<0.05). We note that Pulp Canal Sealer and Endofill sealers decrease cell viability and cellular metabolism when compared to control after 48 hours (p<0.001). MTA and Sealer 26 did not interfere with cell viability in the two evaluation periods (p>0.05). hDPSCs, when grown in the presence of MTA and Sealer 26, express the Nestin, CD105, NANOG and OCT-4 markers, and do not express CD34 and CD45. In turn, MTA and Sealer 26 interfered in the gene expression of DMP1, OC/BGLAP and RUNX2 in relation to the control group (p<0.05), but did not find a significant difference in relation to the ALP gene expression (p>0.05). Therefore, MTA and Sealer 26 demonstrate good biological compatibility when in the presence of hDPSCs. The systematic review showed that almost all materials have good compatibility when in contact with stem cells, being able to be used in clinical practice.

Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies

ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.

Os efeitos da Punica granatum L. em diferentes concentrações sobre duas linhagens celulares: estudo in vitro / The effects of Punica granatum L. at different concentrations on two cell lines: in vitro study

Introdução: A Punica granatum L. (PG), utilizada como medicamento fitoterápico, apresenta propriedades terapêuticas, anti-inflamatórias e antioxidante. Embora diversos estudos já tenham sido realizados com este fitoterápico, seus possíveis efeitos citotóxicos nos tecidos humanos ainda não são claros. Objetivo: Avaliar a citotoxicidade da PG por meio de cultura celular com duas linhagens: fibroblastos humanos de mucosa oral (FLM) e células de carcinoma epidermoide oral humano (KB). Material e método: As células foram submetidas ao teste de viabilidade celular por 24 horas nas concentrações da PG 1%, 0,50%, 0,25%, 0,125%, 0,062% e 0,031%, e aos testes de citotoxicidade celular em 4 horas, 1, 3, 5 e 7 dias, em diferentes concentrações, realizados em triplicata. Foi utilizado um controle negativo (Triton 1%) e um controle positivo sem o extrato de PG. Os dados obtidos foram submetidos à ANOVA (p < 0,05). Resultado: Foi possível observar que o extrato da PG possui efeitos inibitórios, apresentando-se maior nas células KB em relação às FLM. Os testes de citotoxicidade e viabilidade mostraram inibição e morte das células KB e FLM nas concentrações 1%, 0,50% e 0,25%. Conclusão: O efeito inibitório da PG foi dose-dependentes, mostrando-se maior nas células KB em relação às FLM.

Introduction: Punica granatum L. (PG), used as a herbal medicine, has therapeutic, anti-inflammatory and antioxidant properties, although several studies have already been carried out with this herbal medicine, its possible cytotoxic effects on human tissues are still unclear. Objective: To evaluate the cytotoxicity of PG through cell culture with two strains: human oral mucosa fibroblasts (LFM) and human oral squamous cell carcinoma (KB) cells. Material and method: The cells were submitted to the cell viability test for 24 hours in the concentrations of PG 1%, 0.50%, 0.25%, 0.125%, 0.062% and 0.031% and the cell cytotoxicity tests in 4 hours, 1, 3, 5 and 7 days in different concentrations, performed in triplicate. A negative control (Triton 1%) and a positive control without the PG extract were used. The data obtained were submitted to ANOVA (p <0.05). Result: it was possible to observe that the PG extract has inhibitory effects, being higher in KB cells in relation to LFM. The cytotoxicity and viability tests showed inhibition and death of KB and FLM cells at concentrations of 1%, 0.50% and 0.25%. Conclusion: The inhibitory effect of PG was dose dependent, showing itself to be greater in KB cells compared to LMB.

Lipopolysaccharides' Cytotoxicity on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To show the cytotoxicity of Porphyromonas gingivalis lipopolysaccharide (LPS) on human umbilical cord mesenchymal stem cells (HUCMSCs) to better understand the characteristics for its application in regenerative procedures under periodontopathogen LPS influence. Material and Methods: Ultrapure Porphyromonas gingivalis LPS was used in this study. This research used a frozen stock HUCMSCs, previously confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSCs were cultured and divided into two groups, the control group and LPS group with various concentrations from 25 to 0.39 µg/mL. MTT assay was done and the cells were observed and counted. The significance level was set at 5%. Results: The percentage of living HUCMSCs on LPS group were not significantly different among concentrations (p>0.05) from 25 to 0.39 µg/mL, even though there were slight mean decrease between groups, but they were not significant. The duration of 24 hours of exposure of LPS does not significantly lower HUCMSCs viability. Conclusion: LPS does not affect the viability of HUCMSCs. The lower the concentration of LPS, the higher the viability of HUCMSCs.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Phlogiellus bundokalbo spider venom: cytotoxic fractions against human lung adenocarcinoma (A549) cells

Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from Phlogiellus bundokalbo and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells. Methods: Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa FluorTM 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A2 (PLA2) activity of the cytotoxic fractions were also measured. Results: We observed and isolated six fractions from the venom of P. bundokalbo collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA2 activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA2 and cytotoxicity. Conclusion: The venom of P. bundokalbo spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.(AU)

Cytotoxic and anticancer properties of the Malaysian mangrove pit viper (Trimeresurus purpureomaculatus) venom and its disintegrin (purpureomaculin)

The Asiatic pit vipers from the Trimeresurus complex are medically important venomous snakes. These pit vipers are often associated with snakebite that leads to fatal coagulopathy and tissue necrosis. The cytotoxic venoms of Trimeresurus spp.; however, hold great potential for the development of peptide-based anticancer drugs. Methods: This study investigated the cytotoxic effect of the venom from Trimeresurus purpureomaculatus, the mangrove pit viper (also known as shore pit viper) which is native in Malaysia, across a panel of human cancer cell lines from breast, lung, colon and prostate as well as the corresponding normal cell lines of each tissue. Results: The venom exhibited dose-dependent cytotoxic activities on all cell lines tested, with median inhibition concentrations (IC50) ranging from 0.42 to 6.98 µg/mL. The venom has a high selectivity index (SI = 14.54) on breast cancer cell line (MCF7), indicating that it is significantly more cytotoxic toward the cancer than to normal cell lines. Furthermore, the venom was fractionated using C18 reversed-phase high-performance liquid chromatography and the anticancer effect of each protein fraction was examined. Fraction 1 that contains a hydrophilic low molecular weight (approximately 7.5 kDa) protein was found to be the most cytotoxic and selective toward the breast cancer cell line (MCF7). The protein was identified using liquid chromatography-tandem mass spectrometry as a venom disintegrin, termed purpureomaculin in this study. Conclusion: Taken together, the findings revealed the potent and selective cytotoxicity of a disintegrin protein isolated from the Malaysian T. purpureomaculatus venom and suggested its anticancer potential in drug discovery.(AU)

Effects and Mechanism of PARP Inhibitor Olaparib on the Expression of NKG2D Ligands in HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells.@*METHODS@#After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method.@*RESULTS@#10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells.@*CONCLUSION@#Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.

Hallazgos Inmunofenotípicos, Morfológicos y Citogenéticos de las Leucemias Linfoblásticas Agudas en Pediatría / Immunophenotypic, Morphological and Cytogenetic Findings of Acute Lymphoblastic Leukemia in Pediatrics

Introducción: Las leucemias linfoblásticas agudas (LLA) son proliferaciones clónales malignas de células en distintos grados de diferenciación y representan las neoplasias más frecuentes en la edad pediátrica. El objetivo de este estudio es determinar las principales características inmunofenotípicas, biológicas y moleculares de las LLA en nuestro medio. Métodos: Se realiza un estudio retrospectivo, de tipo no experimental, descriptivo, y longitudinal de los pacientes diagnosticados de LLA durante el periodo comprendido entre 2004 a 2009 en el Instituto Oncológico Nacional "Dr. Juan Tanca Marengo" Solca, Guayaquil. Resultados: Se analizaron un total de 316 pacientes, con una edad promedio de 6 años. Por sus características morfológicas fueron clasificados como FAB L1 en 90.5 % de los casos (n=286). En base a su inmunofenotipo 91.8 % (n=290) de los mismos correspondieron a una LLA de fenotipo B y un 8.2 % (n=26) a una de fenotipo T, el con un predominio de la variedad B común. Se reportaron 45 casos de translocaciones, siendo a más común la t(12;21). En relación al cariotipo este se reportó como normal en 229 casos (72.5 %) y se evidenciaron gran variabilidad de alteraciones en el restante 27.5 %, prevaleciendo las hiperdiploidías. Conclusión: Una mejor clasificación y estatificación de los pacientes, por medio de la correlación de los hallazgos inmunofenotípicos, morfológicos y citogenéticos permitirá establecer nuevas estrategias terapéuticas con una mejor sobrevida.

Introduction: Acute lymphoblastic leukemias (ALL) are malignant clonic proliferations of cells at different degrees of differentiation and represent the most frequent neoplasms in pediatric age. The objective of this study is to determine the main immunophenotypic, biological and molecular characteristics of ALL in our environment. Methods: A retrospective, non-experimental, descriptive and longitudinal study of patients diagnosed with ALL during the period between 2004 and 2009 is carried out in the pediatric area of the National Oncology Institute "Dr. Juan Tanca Marengo "Solca, Guayaquil. Results: A total of 316 patients were analyzed, with an average age of 6 years. Due to their morphological characteristics, they were classified as FAB L1 in 90.5% of cases (n = 286). According to their immunophenotype, 91.8% (n = 290) of them corresponded to an ALL of the phenotype B and 8.2% (n = 26) to one of the phenotype T, with a predominance of the common variety B. 45 cases of translocations, the most common being t (12; 21). In relation to the karyotype, this was reported as normal in 229 cases (72.5%) and there was a great variability in the alterations in the rest of 27.5%, with hyperdiploidías prevailing. Conclusion: A better classification and staging of patients, through the correlation of immunophenotypic, morphological and cytogenetic findings, will allow the establishment of new therapeutic strategies with better survival.

Single-cell Analysis of CAR-T Cell Activation Reveals A Mixed T1/T2 Response Independent of Differentiation / 基因组蛋白质组与生物信息学报·英文版

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

High-affinity T cell receptors redirect cytokine-activated T cells (CAT) to kill cancer cells / 医学前沿

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.

Tumor infiltrating T lymphocyte components in malignant pleural effusion of lung adenocarcinoma and their killing activities to autologous tumor cells / 中南大学学报(医学版)

To analyze the components of tumor infiltrating T lymphocyte (TIL) cells in malignant pleural effusion of lung adenocarcinoma, and evaluate their killing activities to autologous tumor cells. 
 Methods: Malignant pleural effusions were collected from 17 patients with lung adenocarcinoma. Mononuclear cells were isolated by Ficoll density gradient centrifugation and flow cytometer was used to analyze TIL cell components. TIL and tumor cells were separated through adherent culture. The tumor cells were identified via intramuscular injection of adherent cells into nude mice and the killing effect of cultured lymphocytes on autologous tumor cells was studied.
 Results: Of the TIL in malignant pleural effusions, T cells accounted for 60.6%-79.3%, while T helper cells were significantly higher than T killer cells (36.63%±1.90% vs 24.64%±2.32%, P<0.001). There were also natural killer (NK) cells and NK T cells in the effusions. Tumor cells were successfully isolated and cultured. The killing activity of cultured TIL to autologous tumor cells was 39.14%±12.04%, and the killing activity of TIL with high proliferation rate to autologous tumor cells was higher than that of low proliferation group (50.51%±3.80% vs 29.04%±5.77%, P<0.001).
 Conclusion: T lymphocytes are the major components of TIL in malignant pleural effusions derived from lung adenocarcinoma, and T helper cells are more than T killer cells. The killing activity of TIL with strong proliferation ability to autologous tumor cells is higher than that of TIL with weak proliferation ability. Therefore, cells from malignant pleural effusions could be used for cellular immunotherapy against tumor.

Biocompatibilidade da resina acrílica de prótese ocular: análise in vitro e in vivo / Biocompatibility of ocular prosthesis acrylic resin: in vitro and in vivo analysis

O objetivo desse estudo foi avaliar a influência de diferentes ciclos e métodos de polimerização da resina acrílica (RA) branca de próteses oculares sobre a biocompatibilidade de células da conjuntiva humana e resposta inflamatória do tecido subcutâneo de ratos. Para isso, foram confeccionados corpos de prova em RA termopolimerizados em água aquecida (RNAA), por energia de microondas (RNTM) e quimicamente ativados (RNQA). Para a análise in vivo, a resposta inflamatória desses 3 grupos (n=20/grupo) foi avaliada no tecido subcutâneo de 20 ratos Wistar por 7, 15, 30 e 60 dias (d). Células inflamatórias foram contadas no tecido adjacente ao corpo de prova após coloração com hematoxilina e eosina. A análise imunohistoquímica foi realizada para a detecção de IL-1ß, IL-6, TNFα, IL-17 e CCL20. Para a análise in vitro, diferentes ciclos de polimerização para cada método citado foram avaliados, totalizando 11 grupos (n=8/grupo). Foram realizadas análises de grau de conversão (GC), MTT, Alamar Blue, ELISA, RT-PCR em tempo real e dupla marcação de Anexina V e iodeto de propídio. Dados quantitativos foram submetidos à Análise de Variância e ao teste de Tukey com significância de 5%. Os resultados qualitativos foram comparados visualmente. Na análise in vivo, houve infiltrado inflamatório moderado para os grupos RNTM e RNQA e leve para o grupo RNAA após 7 d. O infiltrado inflamatório e a imunomarcação dos alvos testados diminuiu gradativamente ao longo dos 60 d. O grupo RNTM exibiu mais células inflamatórias, com exceção do grupo RNAA, que apresentou mais eosinófilos e linfócitos após 15 d, e do grupo RNQA, onde foi observado mais macrófagos em 15 d e neutrófilos em 60 d. Os grupos RNAA e RNQA apresentaram maior imunomarcação de IL-1ß após 7 d. O grupo RNQA apresentou maior imunomarcação de IL-1ß (15 e 30 d), IL-6 (30 e 60 d), IL-17 (15 e 30 d) e TNF-α (7 d). Os grupos RNAA e RNTM apresentaram maior imunomarcação de TNF-α nos períodos de 15 e 30 d, enquanto o grupo RNTM, aos 60 d. Na análise in vitro, todos os grupos apresentaram proliferação celular maior que 75%. O ciclo longo de polimerização em microondas apresentou menor GC e percentual de proliferação celular no MTT e resultou em grande liberação de IL-2. No ensaio de Alamar Blue, esse grupo apresentou baixo percentual de proliferação celular, assim como o grupo que recebeu ciclo longo de polimerização em água aquecida e grupos submetidos à ativação química. Maior liberação de IL-6 foi observada nos grupos submetidos à ativação química e de IL-23 para o ciclo curto de polimerização em microondas. Maior expressão gênica de TGF ß1 ocorreu para o grupo que recebeu ciclo longo de polimerização em água aquecida seguido de 30 min de armazenamento em água. Maior expressão gênica de CASP9 ocorreu para o grupo ativado quimicamente sobre a bancada. Pode-se concluir que os métodos de polimerização por meio de energia de microondas (ciclos longo e curto) e por ativação química desencadearam uma resposta inflamatória mais intensa. Dentre os métodos de polimerização recomendados pelo fabricante, a polimerização em água aquecida apresentou resultados mais satisfatórios(AU)

The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1ß, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1ß immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1ß (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF ß1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results(AU)

Profil clinique et immunologique des patients infectés par le VIH à l'initiation du traitement antirétroviral au centre hospitalier régional de Sokodé (Togo), une décennie après la gratuité de la trithérapie

Introduction : L'initiation tardive du Traitement Anti-Rétroviral (TAR) est fréquente dans les pays à ressources limitées, où la plupart des individus ne connaissent pas leur statut sérologique jusqu'à ce qu'ils soient symptomatiques.Matériel et méthodes : Il s'est agi d'une étude transversale et analytique, qui s'est déroulée du 1er janvier 2016 au 31 décembre 2016 dans le service de médecine générale du Centre Hospitalier Régional (CHR) de Sokodé, le service de référence dans la prise en charge de l'infection à VIH dans la région centrale du Togo.Résultats : Cent-neuf patients dont 15 enfants (13,8%) ont été inclus dans la file active du service de médecine générale du CHR de Sokodé par l'initiation du TAR. La moyenne d'âge des patients était de 32,38 ans [1-59 ans] avec une prédominance féminine (67,9%). La majorité des patients était classée aux stades III (60 cas ; 55%) et au stade IV (11 cas ; 10,1%). Les manifestations cliniques ou affections opportunistes étaient dominées par la diarrhée chronique (15 cas), la fièvre prolongée inexpliquée (12 cas) et la candidose oropharyngée (12 cas). Le taux moyen des CD4 des patients était de 328,54 cellules/mm3 [0-2216 cellules/mm3] et la co-infection VIH-VHB a été retrouvée dans 13,8% (n = 15). L'association Ténofovir (TDF) - Lamivudine (3TC) - Efavirenz (EFV) (87,2% ; n = 95) a été la plus prescrite pour l'initiation du TAR.Conclusion : Le profil des patients infectés par le VIH à l'initiation du TAR à Sokodé est caractérisé par un stade avancé de l'infection à VIH

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. Methods and Results: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~4.4 and molecular mass of 14. 2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1 ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. Conclusions: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.(AU)