Regulatory effect of interleukin-18 on natural killer cell activity in patients with alopecia areata / 中华皮肤科杂志

Objective:To investigate changes of natural killer (NK) cell subsets and interleukin-18 (IL-18) level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity.Methods:A total of 67 patients with alopecia areata (alopecia areata group) and 25 healthy volunteers (control group) were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16 + NK cells. Comparisons between groups were performed using t test or paired t test. Results:Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56 +CD16 - NK cells (8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001) , but significantly increased percentage of CD56 +CD16 + NK cells (46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001) , and there was no significant difference in the percentage of CD56 -CD16 + NK cells between the alopecia areata group and control group (28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383) . The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group (112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015) . After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group (9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively) than in those from the control group (5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05) . After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16 + NK cells (151.10% ± 59.30%) compared with the control group (221.90% ± 93.56%, t = 4.31, P < 0.001) . After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system (14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001) , while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems (both P > 0.05) . Conclusion:IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.

Rice bran hydrolysates induce immunomodulatory effects by suppression of chemotaxis, and modulation of cytokine release and cell-mediated cytotoxicity

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism. Methods: Rice bran hydrolysates were prepared from pigmented rice (Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells (PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells (HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin (PHA)-stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of mTOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Rice bran hydrolysates induce immunomodulatory effects by suppression of chemotaxis, and modulation of cytokine release and cell-mediated cytotoxicity

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism. Methods: Rice bran hydrolysates were prepared from pigmented rice (Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells (PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells (HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin (PHA)- stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of mTOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

MiR-506 Promotes Natural Killer Cell Cytotoxicity against Human Hepatocellular Carcinoma Cells by Targeting STAT3

PURPOSE: It is well documented that natural killer (NK) cytotoxicity against hepatocellular carcinoma (HCC) cells is impaired in HCC, which might account for the failure of anti-tumor immune response. miRNAs are considered as important regulators for the development and functions of NK cells. However, the entire role of miR-506 in NK cells remains far from being addressed. MATERIALS AND METHODS: The expressions of miR-506 and signal transducer and activator of transcription 3 (STAT3) mRNA in primary NK cells from HCC patients and healthy controls were detected by quantitative real-time PCR. NK cell cytotoxicity was assessed by CFSE/7AAD cytotoxicity assay and lactate dehydrogenase assay. Luciferase reporter assay, RNA immunoprecipitation assay, and western blot were conducted to confirm the interaction between miR-506 and STAT3. RESULTS: miR-506 expression was downregulated and STAT3 mRNA was upregulated in primary NK cells from HCC patients. Primary NK cells from HCC patients showed remarkably reduced cytotoxicity against SMMC7721 or HepG2 cells. NK cell cytotoxicity was positively correlated with miR-506 expression and negatively correlated with STAT3 mRNA expression. Additionally, miR-506 overexpression enhanced NK cell cytotoxicity against HCC cells, while miR-506 inhibitor showed the reverse effect. Moreover, miR-506 could suppress STAT3 expression by directly targeting 3′-untranslated regions of STAT3. A negative correlation between miR-506 and STAT3 mRNA expression in HCC patients was observed. Mechanistically, overexpressing STAT3 greatly reversed miR-506-mediated promotion of NK cell cytotoxicity against HCC cells. CONCLUSION: miR-506 enhanced NK cell cytotoxicity against HCC cells by targeting STAT3, suggesting that modulating miR-506 expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

Antineoplastic Effect of Procyanidin-rich Extract of Lafoensia Pacari in Lung Carcinoma Cells

Abstract Lafoensia pacari A. St. Hill has been used in traditional medicine as an anti-ulcerogenic and anti-inflammatory. Although there is an ethnopharmacological indication for cancer treatment, only a few studies have demonstrated its possible anticancer activity. Thus, the aims of this study were: (1) to evaluate the antineoplastic effect of L. pacari ethanolic extract (LPE) in lung carcinoma cells, (2) to determine the mode of action of LPE and (3) to identify the substances present in LPE. Human and murine lung cancer cell lines were grown in vitro and treated with different concentrations of LPE. Cell cycle and caspase-3 activity assays were performed in order to verify the mode of action. LC-ESI-MS screening was performed to detect the compounds present in LPE. LPE showed a dose-dependent cytotoxic effect, where neoplastic cells were more sensitive than non-neoplastic. The LPE induced sub-G1 cell cycle arrest in cancer cells suggesting cell death, which was confirmed as apoptosis by the activation of caspase-3. The LC-ESI-MS analysis indicated a high level of procyanidins, which could be responsible for the antineoplastic effect of LPE. Thus, we concluded that a Lafoensia pacari extract, rich in procyanidins, is cytotoxic against lung cancer cells through activation of caspase-3-dependent apoptosis.

Donor Specific Antibody Negative Antibody-Mediated Rejection after ABO Incompatible Liver Transplantation / 대한이식학회지

Antibody-mediated rejection (AMR) is a major complication after ABO-incompatible liver transplantation. According to the 2016 Banff Working Group on Liver Allograft Criteria for the diagnosis of acute AMR, a positive serum donor specific antibody (DSA) is needed. On the other hand, the clinical significance of the histological findings of AMR in the absence of DSA is unclear. This paper describes a 57-year-old man (blood type, O+) who suffered from hepatitis B virus cirrhosis with hepatocellular carcinoma. Pre-operative DSA and cross-matching were negative. After transplantation, despite the improvement of the liver function, acute AMR was observed in the protocol biopsy on postoperative day 7; the cluster of differentiation 19+ (CD19+) count was 0% and anti-ABO antibody titers were 1:2. This paper presents the allograft injury like AMR in the absence of DSA after ABOi living donor liver transplantation with low titers of anti-ABO antibody and depleted serum CD19+ B cells.

Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.

Engineering of Anti-CD133 Trispecific Molecule Capable of Inducing NK Expansion and Driving Antibody-Dependent Cell-Mediated Cytotoxicity / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The selective elimination of cancer stem cells (CSCs) in tumor patients is a crucial goal because CSCs cause drug refractory relapse. To improve the current conventional bispecific immune-engager platform, a 16133 bispecific natural killer (NK) cell engager (BiKE), consisting of scFvs binding FcγRIII (CD16) on NK cells and CD133 on carcinoma cells, was first synthesized and a modified interleukin (IL)-15 crosslinker capable of stimulating NK effector cells was introduced. MATERIALS AND METHODS: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE). The construct was tested for its specificity using flow cytometry, cytotoxic determinations using chromium release assays, and lytic degranulation. IL-15–mediated expansion was measured using flow-based proliferation assays. The level of interferon (IFN)-γ release was measured because of its importance in the anti-cancer response. RESULTS: 1615133 TriKE induced NK cell–mediated cytotoxicity and NK expansion far greater than that achieved with BiKE devoid of IL-15. The drug binding and induction of cytotoxic degranulation was CD133+ specific and the anti-cancer activity was improved by integrating the IL-15 cross linker. The NK cell–related cytokine release measured by IFN-γ detection was higher than that of BiKE. NK cytokine release studies showed that although the IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18, indicating that release was not at the supraphysiologic level. CONCLUSION: 1615133 TriKE enhances the NK cell anti-cancer activity and provides a self-sustaining mechanism via IL-15 signaling. By improving the NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse mediated by CSCs.

Anti-cancer Effects of a Novel Quinoline Derivative 83b1 on Human Esophageal Squamous Cell Carcinoma through Down-Regulation of COX-2 mRNA and PGE₂ / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: 83b1 is a novel quinoline derivative that has been shown to inhibit cancer growth in human esophageal squamous cell carcinoma (ESCC). This study was conducted to comprehensively evaluate the cytotoxic effects of 83b1 on a series of ESCC cell lines and investigate the mechanisms by which 83b1 suppresses cancer growth based on molecular docking analysis. MATERIALS AND METHODS: A series of ESCC and nontumor immortalized cell lines were exposed to 83b1 and cisplatin (CDDP) in a dose-dependent manner, and the cytotoxicity was examined by a MTS assay kit. Prediction of the molecular targets of 83b1 was conducted by molecular docking analysis. Expression of cyclooxygenase 2 (COX-2) mRNA and COX-2–derived prostaglandin E₂ (PGE₂) were measured by quantitative real-time polymerase chain reaction and enzymelinked immuno-sorbent assay, respectively. In vivo anti-tumor effect was determined using a nude mice xenografted model transplanted with an ESCC cell line, KYSE-450. RESULTS: 83b1 showed the significant anti-cancer effects on all ESCC cell lines compared to CDDP; however, 83b1 revealed much lower toxic effects on non-tumor cell lines than CDDP. The predicted molecular target of 83b1 is peroxisome proliferator-activated receptor delta (PPARδ), which is a widely known oncoprotein. Additionally the expression of COX-2 mRNA and COX-2–derived PGE2 were down-regulated by 83b1 in a dose-dependent manner in ESCC cell lines. Furthermore, 83b1 was shown to significantly reduce the tumor size in nude mice xenograft. CONCLUSION: The results of this study suggest that the potential anti-cancer effects of 83b1 on human esophageal cancers occur through the possible oncotarget, PPARδ, and down-regulation of the cancer related genes and molecules.

Biological characterization of implant surfaces - in vitro study / Caracterização biológica de superfície de titânio - estudo in vitro

Objective: Evaluate the biological performance of titanium alloys grade IV under different surface treatments: sandblasting and double etching (Experimental surface 1; Exp1, NEODENT); surface with wettability increase (Experimental surface 2; Exp2, NEODENT) on response of preliminary differentiation and cell maturation. Material and method: Immortalized osteoblast cells were plated on Exp1 and Exp2 titanium discs. The polystyrene plate surface without disc was used as control group (C). Cell viability was assessed by measuring mitochondrial activity (MTT) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). Data were analyzed using one-way ANOVA and Tukey test. Result: The values of cell viability were: 4h: C - 0.32±0.01A; Exp1 - 0.34±0.08A; Exp2 - 0.29±0.03A. 24h: C - 0.43±0.02A; Exp1 - ; 0.39±0.01A; Exp2 - 0.37±0.03A. The cell adhesion counting was: C -85±10A; Exp1- 35±5B; Exp2& - 20±2B. The amounts of serum total protein were 4d: C - 40±2B; Exp1 - 120±10A; Exp2 -130±20A. 7d: C 38±2B; Exp1 - 75±4A; Exp2 -70±6A. 14 d: C - 100±3A; Exp1 - 130±5A; Exp2 - 137±9A. The values of alkaline phosphatase normalization were: 4d: C - 2.0±0.1C; Exp1 - 5.1±0.8B; Exp2 - 9.8±2.0A<. 7d: C -1.0±0.01C; Exp1 - 5.3±0.5A; Exp2 - 3.0±0.3B. 14 d: C - 4.1±0.3A; Exp1 - 4.4±0.8A; Exp2 - 2.2±0.2B. ...

Objetivo: Avaliar o desempenho biológico de ligas de titânio grau IV submetidos a diferentes tratamentos de superfície - jateamento e duplo ataque ácido (Superfície experimental 1; Exp1, NEODENT) e superfície com aumento na molhabilidade (Superfície experimental 2; Exp2, NEODENT) em resposta preliminar de diferenciação e maturação celular. Material e método: Foram plaqueados osteoblastos imortalizados sobre discos de titânio de Exp1 e Exp2 e como controle o poço da placa de cultura sem disco (C). Empregou-se ensaios de viabilidade celular (MTT) em 4 e 24 horas (n = 5), adesão celular em 4 horas (n = 5), dosagem de proteínas totais e fosfatase alcalina normalizada em 4, 7 e 14 dias (n = 5). Os dados foram analisados por ANOVA em fator único seguido de teste de Tukey. Resultado: Os valores de viabilidade celular foram: 4h: C - 0,32±0,01A; Exp1 - 0,34±0,08A; Exp2 - 0,29±0,03A. 24h: C - 0,43±0,02A; Exp1 - 0,39±0,01A; Exp2 - 0,37±0,03A. A contagem de adesão celular foi: C - 85±10A; Exp1 - 35±5B; Exp2 - 20±2B. Os valores de proteínas totais foram: 4d: C - 40±2B; Exp1 - 120±10A; Exp2 - 130±20A. 7d: C - 38±2B; Exp1 - 75±4A; Exp2 - 70±6A. 14 d: C - 100±3A; Exp1 - 130±5A; Exp2 - 137±9A. Os valores de fosfatase alcalina normalizada foram: 4d: C - 2,0±0,1C; Exp1 - 5,1±0,8B; Exp2 - 9,8±2,0A, 7d: C - 1,0±0,01C; Exp1 - 5,3±0,5A; Exp2 - 3,0±0,3B, 14 d: C - 4,1±0,3A; Exp1 - 4,4±0,8A; Exp2 - 2,2±0,2B. Letras diferentes representam ...

Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function.

Assessment of natural killer cells (NK-cell) cytotoxicity is used not only in research settings but is also important in diagnosis of various diseases. NK-cell cytotoxicity assays are based on measurement of target cells killed by cytotoxic cells analyzed either by chromium (51Cr) release assay or flow cytometry. Both these methods use peripheral blood mononuclear cells (PBMC) or pure NK-cell population and hence require large volume of blood sample which is difficult to obtain in pediatric patients and patients with cytopenia. Hence, a flow cytometric assay was designed to determine NK cell activity using whole blood, eliminating the need for isolation of PBMCs or pure NK cells. This assay is based on a dual fluorescent staining of target cells (K562 cell line). The DIOC18 dye labeled K562 cells are incubated with whole blood and then counterstained with 7-AAD enabling the measurement of dead target cell and then percent cytotoxicity is calculated. This study compared the NK cell cytotoxicity using PBMC and whole blood in clinically relevant samples. There was no significant difference between two assays in the measurement of lytic activity or in reproducibility in the repeated samplings of healthy individuals. The whole blood assay required less volume of blood and also less processing time as compared to PBMC assay. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis expected to have low NK-cell activity. This assay is rapid, sensitive and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK-cell function.

Effects of spermicidal extracts of Ananas comosus (pineapple) and Sapindus saponaria (jaboncillo) on cell viability, cytotoxicity and apoptosis / Efecto del extracto espermicida de Ananas comosus y Sapindus saponaria sobre la viabilidad, la citotoxicidad y la apoptosis celular

Introduction: there are natural products from different fruits and plants that are effective as spermicides, but it is important that they should have little or no cytotoxic effect on epithelial cells. Currently available spermicides with nonoxynol-9 cause vaginal irritation and damage to the vaginal mucosa, the uterine epithelium, and the microbial flora of the vagina. Objective: to elucidate the effect on cell viability, cytotoxicity and apoptosis of spermicidal extracts of Ananas comosus (L.) Merr. and Sapindus saponaria L. over HeLa cell line. Methods: both extracts were evaluated on HeLa cell line using the novel ApoTox-Glo™ Triplex Assay to determine whether cell viability, cytotoxicity and apoptosis were affected. Results: it was determined that treatment with Sapindus saponaria and Ananas comosus extracts initially affected cell viability, but the latter tended to be restored. There was a sign of cell apoptosis that also tended to decrease over time. Conclusions: extracts of Sapindus saponaria and Ananas comosus may affect the survival of cells at the beginning, but these can continue replicating over time. There was a sign of cell apoptosis that also tended to decrease over time. Something similar happened to cell cytotoxicity, indicating that although the extracts may affect the survival of cells at the beginning (6 hours of treatment), these can continue dividing over time.

Introducción: diversos compuestos de procedencia natural como frutos y plantas son altamente efectivos como espermicidas, pero es necesario que estos no tengan efecto citotóxico sobre las células epiteliales. Los espermicidas disponibles actualmente sobre la base de nonoxinol-9, causan irritación y daño en la mucosa, el epitelio uterino y la flora microbiana de la vagina. Objetivo: determinar el efecto sobre la viabilidad, citotoxicidad y apoptosis celular de extractos con actividad espermicida de Ananas comosus (L.) Merr. y Sapindus saponaria L. sobre la línea celular HeLa. Métodos: ambos extractos se evaluaron sobre la línea celular HeLa para determinar el efecto en la viabilidad, la citotoxicidad y la apoptosis celular, utilizando el novedoso ensayo triple ApoTox-Glo™. Resultados: inicialmente el tratamiento con los extractos de Sapindus saponaria y Ananas comosus afectaron la viabilidad celular; sin embargo, esta tendió a restablecerse y mantenerse en el tiempo. Asimismo, la señal de apoptosis celular tendió a disminuir a través de los tiempos de tratamiento. Conclusiones: los extractos de Sapindus saponaria y Ananas comosus podrían afectar la viabilidad celular inicialmente; sin embargo, estas continúan incrementándose con el paso del tiempo. Al mismo tiempo la señal de apoptosis celular disminuyó a través del tiempo y algo similar sucedió con la citotoxicidad celular, indicando que con el paso de las horas los extractos pueden afectar la proliferación celular al inicio (6 h de tratamiento), pero continúan proliferando.

Cytotoxicity of root canal sealers on endothelial cell cultures

This study evaluated, in vitro, the cytotoxicity of six root canal sealers after 12, 24 and 72 h of contact time, using an endothelial ECV-304 cell line. The MTT assay was used for analysis of cell viability. Twelve specimens of each sealer were prepared and randomly assigned to 6 groups according to the commercial brands (n=4/time). A control group was also formed, which was not subjected to the contact with sealers. To assess the effects of sealers on endothelial cells, the specimens were placed in culture plate wells and incubated at 37°C with 5% CO2 and 100% humidity. MTT assays were performed in quadruplicate after 12, 24 and 72 h of contact of the sealer specimens with monolayers. Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test at a significance level of 5%. Analysis of absorbance in the experimental groups showed that GuttaFlow presented the lowest cytotoxicity, with a mean absorbance of 0.048, followed by Pulp Canal Sealer (0.038), Sealer 26 (0.038), Endo Densell (0.036) and Pulp Fill (0.035). The control group had a mean absorbance of 0.098. Based on the results, Endofill and GuttaFlow were the most and the least cytotoxic sealers, respectively.

Este estudo avaliou, in vitro, a citotoxicidade de 6 cimentos endodônticos após 12, 24 e 72 h de tempo de contato, utilizando-se uma linhagem de células endoteliais ECV-304. Para a avaliação da viabilidade celular, utilizou-se o teste de citotoxicidade MTT. Para cada cimento foram preparados 12 corpos de prova que foram distribuídos em 6 grupos experimentais de acordo com as marcas comerciais, sendo 4 para cada tempo. Foi criado um grupo controle que não foi submetido à ação de cimento. Para avaliação do efeito dos cimentos sobre as células endoteliais, os corpos de prova foram inseridos nos poços da placa cultura, incubados a 37°C em presença de 5% de CO2 e 100% de umidade. Os testes MTT foram realizados em quadruplicata, após 12, 24 e 72 h de contato das amostras com o tapete celular. Foi utilizada a prova two-way Anova com o teste post hoc de Bonferroni com nível de significância de 5%. Quando analisadas as médias gerais de absorbância dos grupos analisados observou-se que o cimento GuttaFlow se apresentou como o cimento com menor índice de citotoxicidade, apresentando média de absorbência de 0,048. Logo após, apresentando médias de absorbância iguais (0,038) encontraram-se os cimentos Pulp Canal Sealer e Sealer 26; seguidos do Densell Endo e do Pulp Fill, com 0,036 e 0,035, respectivamente. O grupo controle apresentou média de absorbância de 0,098. Portanto, tendo como base os resultados obtidos, pôde-se concluir que o cimento Endofill foi o que apresentou maior citotoxicidade e o cimento GuttaFlow, o menos citotóxico.

Influence of serum complement and IgG on rituximab-dependent NK cell-mediated cytotoxicity to Raji cells / 白血病·淋巴瘤

Objective To determine the influence of serum complement and IgG on rituximabdependent NK cell-mediated cytotoxicity to Raji cells in vitro.Methods FcγR Ⅲ a (CD16a) polymorphism of NK cells were detected by nest-PCR. Effects of serum IgG on FcγRⅢ a expression of NK cells in vitro were analyzed by flow cytometry.The target cells(Raji cells) were stained with DIO,cultured with effector cells(NK cells) and rituximab with or without serum IgG/complement,and finally stained with propidium iodide (PI),then these cells were tested by flow cytometry and the cytotoxic index was calculated as well. Results The cytotoxic indexes of the ADCC +CDC groups were higher than those of ADCC groups, but the serum IgG groups were lower than the ADCC groups. In FcγRⅢa-158Ⅴ/Ⅴ groups, the cytotoxic indexs of the ADCC+ CDC groups,the serum IgG groups and the ADCC groups were (94.25±1.79) ％,(59.79±0.66) ％ and(69.05± 2.38) ％,respectively,and the differences among the groups were statistically significant (P＜ 0.05).In FcγRⅢ a-158Ⅴ/F groups,the cytotoxic indexs of these three groups were (66.71±5.57) ％,(18.13±2.99) ％ and (39.63±3.86) ％, respectively, and the differences among the groups were also statistically significant (P＜ 0.05).Conclusions Complement may enhance the rituximab-mediated NK cell cytotoxicity to Raji cells, whereas,serum IgG may weaken the cytotoxicity against Raji cells. It is clued up that for patients treated by tumorspecific monolonal antibody (MAb), combined infusion of fresh frozen plasma could promote its anti-tumor effect,however,MAb combined with IVIG may impair its anti-tumor effect.

Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells

This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (p<0.05). Statistically significant differences (p<0.05) were observed between the membranes and the control in the experimental period, although there was a significant reduction in viability over time (p<0.01). At 4 and 12 h, the porcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (p<0.01), and this difference was maintained at 24 h (p<0.05). This in vitro study showed that the porcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time.

Neste estudo foi comparada a citoxicidade e a liberação de nitrito induzidos por membranas de colágeno bovino e suíno em células mononucleares humanas. Foram coletados sangue periférico de cada paciente, e realizada separação de mononucleares por gradiente de Ficoll. Um total de 2x10(5) células foram plaqueadas em placas de cultura de 48 poços sob as membranas, em triplicata. O poço sem membrana serviu como controle negativo. A viabilidade celular foi avaliada medindo a atividade mitocondrial (MTT) em 4,12 e 24 h, com dosagens dos níveis de nitrito pelo método de Griess nos mesmos períodos. As amostras não apresentaram distribuição normal, sendo realizado o teste de Kruskal-Wallis (p<0,05). Foram observadas diferenças estatisticamente significantes entre as membranas e o controle nos período analisados (p<0,05), embora tenha ocorrido redução da viabilidade em função do tempo (p<0,01). Em 4 e 12 h a membrana suína induziu maior liberação de nitrito comparado ao controle e à membrana bovina, respectivamente (p<0,01). Tal diferença foi mantida em 24 h (p<0,05). Este estudo in vitro demonstrou que a membrana colágena suína induz uma maior produção de mediador pró-inflamatório pelas células mononucleares nas primeiras horas de contato, diminuindo com o tempo.

Mechanisms involved in natural killer cell mediated target cell death leading to spontaneous tumour regression.

We have studied the mechanisms involved in the spontaneous regression of a rat histiocytoma in syngeneic hosts and tumour cell death processes. In addition to the natural killer (NK) cells which act through antibody dependent cellular cytotoxicity (ADCC), TNF-α also participates in the induction of necrosis in tumours. We have shown that the tumour cell is killed by necrosis which is perforce mediated, and apoptosis leading to target cell DNA fragmentation. A prior activation of the effector cells is essential before it can kill the target cell, as naive effector cells are ineffective. Activation of effector cells is mediated by Thl type of cytokines in viro and in vivo. IFN-γ seems to play an important role in tumour regression as injection of antibodies to IFN- γ in vivo inhibited tumour rejection.

The effect of recombinant human erythropoietin(rHuEPO) on NK cell cytotoxicity in hemodialysis patients / 中华肾脏病杂志

Objective To evaluate the relationship between NK cytotoxicity and anemia in uremia. Methods The effect of rHuEPO on natural killer (NK) cell cytotoxic activity was studied in 12 hemodialysis(HD) patients. Results The levels of hemoglobin (Hb) and NK cell activity were significantly lower in HID patients than that in healthy controls. After two months of the treatment with rHuEPO, the levels of Hb in these patients rose significantly with a parallel rise in NK cell activity. NK cell activity was not increased when they were incubated with rHuEOP but was increased with red blcxxl cells. Conclusion Improved NK cell cytotoxicity in HD patients after treatment with rHuEPO was achieved through the rise in R15C rather than through rHuEPO itself.

Natural killer activity and antibody-dependent cellular cytotoxicity in patients with primary lung cancer

The NK activity and ADCC of peripheral blood mononuclear cell were examined to evaluate the contribution of ADCC and NK activity to host immune response against lung cancer. The NK activity and ADCC were examined in 58 patients with primary lung cancer and 40 healthy volunteers as normal controls. The NK activity of patients with lung cancer was significantly subnormal, but ADCC was at a normal level. The NK activity was decreased in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC) compared to normal controls. According to stage, the NK activity in stage II, III-M0 and III-M1 NSCLC showed low levels compared to that of stage I NSCLC, but there was no difference of NK activity in patients with SCLC. The NK activity was not affected by performance status. There was no significant difference of ADCC in patients with lung cancer according to cell type, stage and performance compared with that of normal controls. The NK activity and ADCC were not changed after chemotherapy and operation respectively.

Natural Killer Cell Activity in Rheumatoid Arthritis Measured by a Single Cell Cytotoxicity Assay

The natural killer(NK) cell activity of mononuclear cells (MNC) from peripheral blood (PB) and synovial fluid (SF) of 40 rheumatoid arthritis(RA) patients was investigated by employing 51-chromium-(51Cr) release microcytotoxicity and single cell cytotoxicity assays against K562 target cells. It has been revealed that SF-MNC from RA patients showed a significantly lower NK activity than PB-MNC from the same patients and this might be due to an impaired target binding capacity of the effector cells and not due to a deficiency of active NK cells.

The enhancing effect of human interleukin-18 expressed in nasopharyngeal carcino-ma cells on the cytotoxicity of CD8+ T cells from patient with nasopharyngeal carci-noma / 中国免疫学杂志

The study is designed to investigate if human interleukin 18 (hIL-18) cDNA expressed in nasopharyngeal carcinoma (NPC) cells could enhance the cytotoxicity of CD8+ T cells from the PBMC of the MFC-bearing patient and how to enhance it.Methods: A constructed eukaryotic expression vector for human IL-18 ,pcDNA3.1(-)/hIL-18, was used to transfect the NPC cell strain-SUNE cells. Then, the transfected SUNE cells were mix-cultured with CD8+ T cells which were separated from the PBMC of the patient with NPC, the cytotoxicity was measured by lactate dehydrogenase(LDH) releasing method, the expression for Perforin, Fas-L and Granzyme B were detected with the mix-cultures. Results: The eukaryotic expression vector could highly express hIL-18 in transfected SUNE cells and only traces of the protein exist in non-transfected SUNE cells. The concentration of hIL-18 in supernatant of the transfected SUNE cells was (85 ? 10) pg/ml, but less than 5 pg/ml of hIL-18 contained in supernatant of the non-transfected SUNE cells.The expression of hIL-18 in SUNE cells could enhance the cytotoxicity of CD8+ T cells from the NPC patient significantly( P