Synergistic antimicrobial potential of EGCG and fosfomycin against biofilms associated with endodontic infections

Abstract Objective This study aimed to evaluate the cytotoxicity and synergistic effect of epigallocatechin gallate (EGCG) and fosfomycin (FOSFO) on biofilms of oral bacteria associated with endodontic infections. Methodology This study determined minimum inhibitory and bactericidal concentration (MIC/MBC) and fractionated inhibitory concentration (FIC) of EGCG and FOSFO against Enterococcus faecalis, Actinomyces israelii, Streptococcus mutans, and Fusobacterium nucleatum. Monospecies and multispecies biofilms with those bacteria formed in polystyrene microplates and in radicular dentin blocks of bovine teeth were treated with the compounds and control chlorhexidine (CHX) and evaluated by bacterial counts and microscopy analysis. Toxicity effect of the compounds was determined on fibroblasts culture by methyl tetrazolium assays. Results The combination of EGCG + FOSFO demonstrated synergism against all bacterial species, with an FIC index ranging from 0.35 to 0.5. At the MIC/FIC concentrations, EGCG, FOSFO, and EGCG+FOSFO were not toxic to fibroblasts. EGCG+FOSFO significantly reduced monospecies biofilms of E. faecalis and A. israelli, whereas S. mutans and F. nucleatum biofilms were eliminated by all compounds. Scanning electron microscopy of multispecies biofilms treated with EGCG, EGCG+FOSFO, and CHX at 100x MIC showed evident biofilm disorganization and substantial reduction of extracellular matrix. Confocal microscopy observed a significant reduction of multispecies biofilms formed in dentin tubules with 84.85%, 78.49%, and 50.6% of dead cells for EGCG+FOSFO, EGCG, and CHX at 100x MIC, respectively. Conclusion EGCG and fosfomycin showed a synergistic effect against biofilms of oral pathogens related to root canal infections without causing cytotoxicity.

Exposure to lipopolysaccharide and calcium silicate-based materials affects the behavior of dental pulp cells

Abstract This study assessed the cell viability, cytokine production, and mineralization potential of human dental pulp cells (hDPCs) after exposure to lipopolysaccharide (LPS) and application of calcium silicate-based materials (CSBM). Characterization of the CSBM was performed by infrared spectroscopy (n = 3). Extracts of Bio-C Repair, Biodentine, Cimmo HD, and MTA Repair HP were prepared and diluted (1:1, 1:4, and 1:16). Culture of hDPCs was established and treated or not with 1 µg/mL of LPS from Escherichia coli for 7 days. MTT assay was used to assess cell viability at 24, 48, and 72 h (n = 6). Alkaline phosphatase (ALP) activity was assayed on day 7 (n = 4). Il-10 and TNF-α were quantified by ELISA at 24 h (n = 6). Data were analyzed by ANOVA and Tukey's test (α = 0.05). Cell viability of LPS-activated hPDCs was higher than untreated control in 48 and 72 h (p < 0.05). Differences between non-treated and LPS-activated hPDCs were observed for Biodentine and Cimmo HP (p < 0.05). The CSBM influenced the cell viability (p < 0.05). ALP activity was higher in LPS-activated hDPCs (p < 0.05). No changes in the concentration of TNF-α were observed between groups (p > 0.05). The CSBM increased the Il-10 production (p < 0.05). LPS-activated hDPCs presented increased cell viability and ALP activity. The CSBM showed mild toxicity and was able to enhance the cell viability and mineralization potential of untreated and LPS-activated hDPCs. The CSBM also induced anti-inflammatory mechanisms without compromising pro-inflammatory ones.

Resumo Este estudo avaliou a viabilidade celular, produção de citocinas e potencial de mineralização de células da polpa dentária humana (hDPCs) após exposição a lipopolissacarídeo (LPS) e aplicação de materiais à base de silicato de cálcio (CSBM). A caracterização do CSBM foi realizada por espectroscopia (n = 3). Extratos de Bio-C Repair, Biodentine, Cimmo HD e MTA Repair HP foram preparados e diluídos (1: 1, 1: 4 e 1:16). A cultura de hDPCs foi estabelecida e tratada ou não com 1 µg / mL de LPS de Escherichia coli por 7 dias. O ensaio de MTT foi usado para avaliar a viabilidade celular em 24, 48 e 72 h (n = 6). A atividade da fosfatase alcalina (ALP) foi avaliada no dia 7 (n = 4). Il-10 e TNF-α foram quantificados por ELISA em 24 h (n = 6). Os dados foram analisados ​​por ANOVA e teste de Tukey (α = 0,05). A viabilidade celular das hPDCs ativados por LPS foi maior do que o controle não tratado em 48 e 72 h (p <0,05). Diferenças entre hPDCs não tratados e ativados por LPS foram observados para Biodentine e Cimmo HP (p < 0,05). Os CSBM influenciaram na viabilidade celular (p <0,05). A atividade de ALP foi maior em hDPCs ativadas por LPS (p <0,05). Não foram observadas alterações na concentração de TNF-α entre os grupos (p> 0,05). Os CSBM aumentaram a produção de Il-10 (p < 0,05). Os hDPCs ativados por LPS apresentaram um aumento na viabilidade celular e atividade ALP. Os CSBM apresentaram toxicidade moderada e foram capazes de aumentar a viabilidade celular e o potencial de mineralização de hDPCs não tratados e ativados por LPS. Os CSBM também induziram mecanismos anti-inflamatórios sem comprometer os pró-inflamatórios.

Aspectos técnicos y clínicos de la prueba cruzada de histocompatibilidad en el trasplante de órganos solidos / Technical and clinical aspects of the histocompatibility crossmatch assay in solid organ transplantation

La presencia de anticuerpos dirigidos contra los antígenos leucocitarios humanos (Human Leukocyte Antigens, HLA) que se expresan en las células del donante, es uno de los factores de riesgo más importantes asociados con las complicaciones clínicas después del trasplante. La prueba cruzada es una de las pruebas de histocompatibilidad más eficaces para la detección de anticuerpos específicos contra el donante en los receptores de injertos. En los primeros métodos de la prueba cruzada, se utilizaba la citotoxicidad dependiente del complemento, que es útil para detectar dichos anticuerpos responsables del rechazo hiperagudo del injerto, pero carece de la sensibilidad adecuada. Por ello, se desarrollaron métodos de pruebas cruzadas más sensibles, entre ellas, la prueba cruzada por citometría de flujo que hoy se considera el método preferido. En este artículo se revisa la evolución de la prueba cruzada y los factores más importantes que deben tenerse en cuenta al realizarla y al interpretar los resultados de esta prueba fundamental para la supervivencia a largo plazo del injerto.

The presence of antibodies directed against human leukocyte antigens (HLA) expressed on donor cells is a significant risk factor for serious clinical complications after transplantation. The crossmatch assay is one of the most important tests available for the detection of donor-specific antibodies in potential allograft recipients. Early crossmatch methods utilized complement-dependent cytotoxicity, which is useful for detecting the donor-specific anti- HLA antibodies responsible for hyperacute allograft rejection but lacks adequate sensitivity. Consequently, more sensitive crossmatch methods have been developed, ultimately leading to the flow cytometry crossmatch as the currently preferred methodology. Herein, we review the evolution of the crossmatch assay and the most important factors to consider when performing and interpreting the results of this fundamental assay for ensuring the long-term survival of the transplanted organ.

Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

Calcium silicate-based cements affect the cell viability and the release of TGF-β1 from apical papilla cells

Abstract This study investigated the cytotoxicity and release of Transforming Growth Factor Beta 1 (TGF-β1) from cultured human apical papilla cells (APCs) after application of four bioactive materials. Culture of APCs was established and used for cytotoxic and quantitative assays. Extracts of Biodentine, Bio-C Repair, MTA Repair and White MTA were prepared and diluted (1, 1:4 and 1:16) and used for MTT assays up to 72 h. Total TGF-β1 was quantified by ELISA. Data were analyzed by ANOVA and Tukey's test (α = 0.05). For Biodentine, at 24 h and 48 h, cell viability was lower than control (p < 0.05). At 72 h, only undiluted extract of Biodentine were cytotoxic (p < 0.05). At 24 h, a cytotoxic effect was found for undiluted and 1:4 dilution of Bio-C Repair (p < 0.05). At 48 h, however, Bio-C Repair at 1:4 and 1:8 dilution showed higher cell viability (p < 0.05). At 24 and 48 h, the cell viability for undiluted MTA Repair were higher than control (p < 0.05). For White MTA, at 24 and 48 h, all dilutions were cytotoxic (p < 0.05). All cements led to reduced release of total TGF-β1 from the APCs (p < 0.05). In conclusion, cell viability varied depending on the material and dilution. Only Bio-C repair and MTA repair led to higher cell viability of APCs. All materials induced a decrease in the release of total TGF-β1 from the APCs.

Resumo Este estudo investigou a citotoxicidade e liberação do Fator de Crescimento Transformador Beta 1 (TGF-β1) em células da papila apical humana (APCs) cultivadas após a aplicação de quatro materiais bioativos. A cultura de APCs foi estabelecida e usada para ensaios citotóxicos e quantitativos. Extratos de Biodentine, Bio-C Repair, MTA Repair e White MTA foram preparados e diluídos (1, 1: 4 e 1:16) e usados para ensaios de MTT por até 72 h. O TGF-β1 total foi quantificado por ELISA. Os dados foram analisados por ANOVA e teste de Tukey (α = 0,05). Para o Biodentine, em 24 h e 48 h, efeito citotóxico foi observado (p <0,05). Em 72 h, apenas o extrato não diluído de Biodentine teve efeito citotóxico (p <0,05). Em 24 h, valores mais baixos de viabilidade celular foram encontrados para o extrato não diluído e diluidi 1:4 de Bio-C Repair (p <0,05). Em 48 h, no entanto, Bio-C Repair na diluição 1:4 e 1:8 mostrou maior viabilidade celular (p <0,05). A viabilidade celular para MTA Repair não diluído em 24 e 48 h foi maior que o controle (p <0,05). Para White MTA, às 24 e 48 h, a viabilidade celular em todas as diluições foram citotóxicas (p <0,05). Todos os cimentos levaram à redução da liberação de TGF-β1 total das APCs (p <0,05). Em conclusão, a viabilidade celular variou dependendo do material e da diluição. Biodentine, Bio-C Repair e MTA Repair levaram a uma maior viabilidade celular de APCs. Todos os materiais induziram uma diminuição na liberação de TGF-β1 total das APCs.

Phasing out the pre-transplant cytotoxicity crossmatch: Are we missing something? / Exclusão da prova cruzada por citotoxicidade pré-transplante: estamos perdendo algo

Abstract Introduction: The anti-human globulin-enhanced complement-dependent cytotoxicity crossmatch (AHG-CDCXM) assay has been used to assess the presence of donor-specific antibodies (DSA) in recipient's serum before kidney transplantation. The flow cytometric crossmatch (FCXM) assay was first introduced as an additional test. The aim of this study was to clinically validate the single use of the FCXM assay. Methods: This study compared the outcomes of a cohort of kidney transplant patients that underwent FCXM only (FCXM group) versus a cohort of kidney transplant patients that underwent AHG-CDCXM (control group). Results: Ninety-seven patients in the FCXM group and 98 controls were included. All crossmatches in the control group were negative. One patient in the FCXM group had a positive B cell crossmatch. One year after transplantation, there were no significant differences in patient survival (p = 0.591) and graft survival (p = 0.692) between the groups. Also, no significant difference was found in the incidence of Banff ≥ 1A acute cellular rejection episodes (p = 0.289). However, acute antibody-mediated rejections occurred in 3 controls (p = 0.028). Conclusion: The results showed that discontinuing the AHG-CDCXM assay does not modify the clinical outcomes in a 1-year follow-up.

Resumo Introdução: O ensaio de prova cruzada por citotoxicidade dependente do complemento antiglobulina humana (AHG-CDCXM - do inglês anti-human globulin-enhanced complement-dependent cytotoxicity crossmatch) tem sido usado para avaliar a presença de anticorpos específicos contra o doador (DSA - do inglês donor-specific antibodies) no soro do receptor antes do transplante renal. O ensaio de prova cruzada por citometria de fluxo (CFXM) foi introduzido pela primeira vez como um teste adicional. O objetivo deste estudo foi validar clinicamente o uso único do ensaio CFXM. Métodos: Este estudo comparou os resultados de uma coorte de pacientes de transplante renal que foram submetidos apenas ao CFXM (grupo CFXM) contra uma coorte de pacientes de transplante renal submetidos ao AHG-CDCXM (grupo controle). Resultados: Foram incluídos noventa e sete pacientes no grupo CFXM e 98 controles. Todas as provas cruzadas no grupo controle foram negativas. Um paciente no grupo CFXM teve uma prova cruzada positiva para células B. Um ano após o transplante, não houve diferenças significativas na sobrevida do paciente (p = 0,591) e na sobrevida do enxerto (p = 0,692) entre os grupos. Também não foi encontrada diferença significativa na incidência de episódios de rejeição aguda celular (p = 0,289) segundo critério de Banff ≥ 1A. No entanto, rejeições agudas mediadas por anticorpos ocorreram em 3 controles (p = 0,028). Conclusão: Os resultados mostraram que a interrupção do ensaio AHG-CDCXM não modifica os desfechos clínicos em um acompanhamento de 1 ano.

Evaluation of the viability of BEAS-2B cells exposed to gasoline engine exhaust with different particle sizes by air-liquid interface / 中华劳动卫生职业病杂志

Objective@#To evaluate the viability of gasoline engine exhaust (GEE) with different particle sizes on human lung cell line BEAS-2B in vitro by air-liquid interface (ALI) .@*Methods@#GEE were collected with a Tedlar bag and their particulate matter (PM) number, surface and mass concentration in three kind of GEE (filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter) were measured by two type of particle size spectrometer including TSI-3321 and SMPS-3938. Five groups were included, which divided into blank control group, clean air group, filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group. Except the blank control group, BEAS-2B cells, cultured on the surface of Transwells, were treated with clean air or GEE by ALI method at a flow rate of 25 ml/min, 37 ℃ for 60 min in vitro. CCK-8 cytotoxicity test kit was used to determine the cell relative viability of BEAS-2B cells.@*Results@#In the filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter, high concentrations of fine particles can be detected, but the coarse particles only accounted for a small proportion, and the sequence of PM concentration was motorcycle exhaust without three-way catalytic converter group> non-filtered automobile exhaust group> filtered automobile exhaust group (P<0.001) . Compared with the clean air group, the cell relative viability in the 3 GEE-exposed groups were significantly lower (P<0.001) . Among the comparisons of GEE exposure groups with different particle size spectra, the sequence of the cell relative viability was filtered automobile exhaust group >non-filtered automobile exhaust group> motorcycle exhaust without three-way catalytic converter group (P<0.001) . When took the clean air control group as a reference, the mean of the cell relative viability in the filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group, was decreased by 26.34%, 36.00% and 49.59%, respectively.@*Conclusion@#GEE with different particle size spectra could induce different levels of toxic effects to the human lung cells BEAS-2B by ALI. After lowering the concentration of particles in the GEE and using the three-way catalytic converter could obviously improve the survival rate of lung cells.

Assessment of intracanal medications cytotoxicity on L929 fibroblast cells / Avaliação da citotoxicidade de medicamentos intracanais em fibroblastos L929

This study aimed to evaluate the cytotoxicity of intracanal medications on L929 fibroblast cells at different periods of observation. The following experimental groups were studied: calcium hydroxide with camphorated paramonochlorophenol and glycerin (CPG); iodoform with glycerin (IG); calcium hydroxide with iodoform and distilled water (CIW); iodoform with distilled water (IW); calcium hydroxide with distilled water (CW); Otosporin ® (OT); and a control group composed of cells and culture medium. Eluates were prepared from each group and placed in contact with 1 x 105 cells/well for periods of 30 minutes, 12, 24, 48 and 72 hours, 5 and 7 days. After each experimental period, a cytotoxicity test was performed using methyltetrazolium (MTT) and a spectrophotometer at an optical density of 570 nm to analyze cell viability. The ANOVA and Tukey test with a significance level of 5% was used to analyze the data. At 30 minutes and at 12 hours, all groups were equal to the control group. At 24 hours, there was greater cytotoxicity in the IG group than in the control group (P<0.001). At 48 hours, only the OT group was cytotoxic (P <0.001). At 72 hours and at 5 days, the most cytotoxic groups were CW and OT. At 7 days, the IW and CPG groups were the least cytotoxic (P <0.001). With respect to experimental time, significant differences between 24 hours and 7 days were observed in all groups. Otosporin® was the most cytotoxic medication, followed by calcium hydroxide with distilled water.

Este estudo teve como objetivo avaliar a citotoxicidade de medicações intracanais em células L929 de fibroblastos em diferentes períodos de observação. Os seguintes grupos experimentais foram estudados: hidróxido de cálcio com paramonoclorofenol canforado e glicerina (CPG); iodofórmio com glicerina (IG); hidróxido de cálcio com iodofórmio e água destilada (CIW); iodofórmio com água destilada (IW); hidróxido de cálcio com água destilada (CW); Otosporin® (OT); e um grupo controle composto por células e meio de cultura. Os eluatos foram preparados a partir de cada grupo e colocados em contato com 1 x 105 células/poço, por períodos de 30 minutos, 12, 24, 48 e 72 horas, 5 e 7 dias. Depois de cada período experimental, um teste de citotoxicidade foi realizado utilizando metiltetrazólio (MTT) e um espectrofotômetro a uma densidade óptica de 570 nm para analisar a viabilidade celular. A análise de variância e o teste de Tukey com nível de significância de 5% foi utilizado para analisar os dados. Em 30 minutos e em 12 horas, todos os grupos foram iguais ao grupo controle. Em 24 horas, houve uma maior citotoxicidade no grupo IG do que no grupo controle (P<0,001). Em 48 horas, apenas o grupo OT foi citotóxico (P<0,001). Em 72 horas e em 5 dias, os grupos mais citotóxicos foram CW e OT. Aos 7 dias, os grupos IW e CPG foram os menos citotóxicos (P<0,001). Com relação ao tempo experimental, foram observadas diferenças significativas entre 24 horas e 7 dias em todos os grupos. Conclusão: Otosporin® foi o medicamento mais citotóxico, seguido de hidróxido de cálcio com água destilada.

Effect of myrrh and thyme on Trichinella spiralisenteral and parenteral phases with inducible nitric oxide expression in mice

Trichinellosis is a serious disease with no satisfactory treatment. We aimed to assess the effect of myrrh (Commiphora molmol) and, for the first time, thyme (Thymus vulgaris L.) against enteral and encysted (parenteral) phases of Trichinella spiralis in mice compared with albendazole, and detect their effect on inducible nitric oxide synthase (iNOS) expression. Oral administration of 500 mg/kg of myrrh and thyme led to adult reduction (90.9%, 79.4%), while 1,000 mg/kg led to larvae reduction (79.6%, 71.3%), respectively. Administration of 50 mg/kg of albendazole resulted in adult and larvae reduction (94.2%, 90.9%). Positive immunostaining of inflammatory cells infiltrating intestinal mucosa and submucosa of all treated groups was detected. Myrrh-treated mice showed the highest iNOS expression followed by albendazole, then thyme. On the other hand, both myrrh and thyme-treated groups showed stronger iNOS expression of inflammatory cells infiltrating and surrounding encapsulated T. spiralis larvae than albendazole treated group. In conclusion, myrrh and thyme extracts are highly effective against both phases of T. spiralis and showed strong iNOS expressions, especially myrrh which could be a promising alternative drug. This experiment provides a basis for further exploration of this plant by isolation and retesting the active principles of both extracts against different stages of T. spiralis.

Analysis of platelet eluate for the elucidation of sensitization to HLA in kidney transplant candidate / Análise do eluato de plaqueta para a elucidação da sensibilização ao HLA em um candidato ao transplante de rim

While a 42-year-old male patient was being prepared for deceased-donor renal transplantation, anti-HLA-A2 antibodies were detected in the serum by enzyme-linked immunosorbent assay (ELISA) method. The patient denied any transfusion history and previous transplant. Crossmatch by complement dependent cytotoxicity (CDC) and CDC with anti -human globulin (CDC-AHG) proved negative with a four-cell panel with positive typing for HLA-A2. Adsorption of antibodies with platelets and analysis of eluate were suggested to elucidate discrepancies in results by ELISA and by CDC-AHG. ELISA showed that adsorbed serum with platelets did not reveal antibodies for HLA-A2 specificity and suggested that they were removed by their specific binding with HLA-A2 antigens on the platelet surface. Eluate analysis by ELISA showed antibodies for HLA-A2 specificity. No antibodies for HLA-A2 specificity in the non-adsorbed serum were detected by CDC-AHG method. Revision of patient's data showed that a previous transfusion had occurred, which may have been the source of HLA sensitization. The suggested method may be a contribution towards the evaluation of sensitivity between CDC-AHG and ELISA methods for characterizing antibodies in the patient's serum.

Enquanto um paciente do sexo masculino de 42 anos de idade estava sendo preparado para o transplante renal de doador falecido, anticorpos anti-HLA-A2 foram detectados no soro pelo método de ensaio imunoenzimático (ELISA). O paciente negava história de transfusão e transplante anterior. Prova-cruzada por citotoxicidade dependente de complemento (CDC) e CDC com antiglobulina humana (CDC-AGH) foram negativos com um painel de quatro células com tipagem positiva para HLA-A2. O método de adsorção de anticorpos com plaquetas e análise do eluato foi sugerido para explicar as discrepâncias dos resultados de ELISA e CDC-AGH. O método de ELISA mostrou que o soro adsorvido com plaquetas não revelou anticorpos para especificidade HLA-A2, sugerindo que eles foram removidos por meio de sua ligação específica com os antígenos HLA-A2 na superfície das plaquetas. A análise do eluato por ELISA mostrou anticorpos para especificidade HLA-A2. Nenhum anticorpo para especificidade HLA-A2 foi detectada no soro não adsorvido pelo método de CDC-AGH. Revisão dos dados do paciente mostrou que houve transfusão anterior, podendo ter sido a fonte de sensibilização HLA. O método sugerido é uma contribuição para avaliação da sensibilidade entre os métodos de CDC-AGH e ELISA em caracterizar anticorpos no soro do paciente.

Dentin barrier cytotoxicity test with three-dimensional cell cultures / 北京大学学报(医学版)

Objective:To evaluate the cytotoxicity of four dentin filling materials and two dentin adhe-sives with a dentin barrier test and to compare the results with those in a conventional filter diffusion test in order to investigate the advantages of the dentin barrier test.Methods: Eugenol cement, zinc phos-phate cement, adhesive glass ionomer cement, composite resin and two self-etching adhesives ( REMI BOND and Adper Easy One) were tested.In the dentin barrier test, L929 mouse fibroblasts were three-dimensional cultured in polystyrene meshes.The dentin disks were cut from the human third molars, near the pulp and in parallel with the occlusal surface, and their permeability within the measurement area was evaluated by a hydraulic permeability device.A mesh with the cells was placed in the “pulp cavity” of the chamber and one dentin disk was put on the cell mesh and its “pulp side” was in contact with the mesh.The test materials and controls were in contact with the“occlusal side” of the dentine disks for 24 h.The cell viability was obtained with MTT assay and the results were expressed as a percentage of con-trol tissues.The Mann-Whitney U test was used to make the statistical analyses.In the filter diffusion test, after a 24 h contact between the test materials and the filters with monolayer cells, the filters were dyed and the grades of cytotoxicity were decided.Results:A mean permeability of the dentin disks near the pulp was 0.293 μL/(min· cm2· cmH2O)(1 cmH2O=0.098 kPa);In the dentin barrier test, Eu-genol cement, REMI BOND and Adper Easy One respectively reduced the cell survival rates to 82%, 63%and 54%.Other materials showed no or very low toxic reactions; In the filter diffusion test, the light-curing composite resin was moderately cytotoxic, the dental adhesive glass ionomer cement was mild-ly cytotoxic and the others were severely cytotoxic;All the six materials in the dentin barrier test had low-er cytotoxicity than in the filter diffusion test.Conclusion:The cytotoxicity of the test materials using the dentin barrier test with three-dimensional cell cultures is lower than that in the filter diffusion test, which has good correlation with the clinical situation.

The efficiency and function detection of NK cell differentiation from human umbilical cord hematopoietic stem cells in vitro / 天津医药

Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord he?matopoietic stem cells (HSCs) in vitro. Methods CD34+hematopoietic stem cells were isolated from human umbilical cord blood, and inoculated into SCGM medium containing 20 μg/L FMS like tyrosine kinase 3 ligand (Flt-3L), stem cell fac?tor (SCF), interleukin (IL)-7, IL-15 and IL-21. And CD34+HSCs were differentiated into NK cells in directional inducing. The growth state of cells was observed. The expressions of CD56, NKG2D, NKp46, CD3, CD19 and CD34 were detected by flow cytometry in the differentiation of 7, 14, 21 and 28 d. In the differentiation of 21 d and 28 d, the differentiation cells were used as effector cells, and K562 cells as target cells. The ratios of effector cells and target cells were 8∶1, 4∶1, 2∶1 and 1∶1. The killing activity of the differentiated cells was detected by lactate dehydrogenase (LDH) cell toxicity assay and 7AAD/CFSE labeling method. Results CD34+HSCs derived from human umbilical cord blood can proliferate in vitro under appropriate condition. There were no significant differences in the expression of CD3 and CD19 between different differentia?tion stages (7, 14, 21 and 28 d, P>0.05). The expressions of CD56, NKG2D and NKp46 were significantly different (P<0.05), and the ultimate expression amount was (72.57±1.60)%, (32.83±1.29)%and (29.53±2.40)%. The expression of CD34 decreased gradually, and the lowest was (12.13 ± 2.01)%. The maximum killing activity detected by LDH cell toxicity assay and 7AAD/CFSE labeling method reached(49.91±2.76)%and (40.87±1.12)%.The killing activity of NK cells was decreased in the order of 8∶1, 4∶1, 2∶1 and 1∶1 groups (P<0.05). There was no significant difference in the killing activity between NK cells of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem cells can differentiate into NK cells un? der appropriate conditions in vitro, and the NK cells induced from differentiation are with killing activity.

Variaciones en el número y función de los linfocitos asesinos naturales durante infecciones recurrentes o graves / Variation in NK cell number and function in individuals with recurrent or severe infections

Introducción. Existen pocos datos sobre los defectos que afectan el desarrollo y función de los linfocitos asesinos naturales ( natural killers, NK) en pacientes con un incremento anormal en la recurrencia de infecciones. Objetivo. Realizar una evaluación sistemática de las diferentes subpoblaciones y la función de estas células en pacientes con infecciones recurrentes. Materiales y métodos. Se incluyeron 20 pacientes con infecciones graves o recurrentes y se analizaron las subpoblaciones y la respuesta citotóxica de los linfocitos NK en sangre periférica. Los resultados de los pacientes se compararon con controles sanos pareados por edad y sexo. Resultados. Los pacientes con episodios infecciosos activos presentaron anormalidades transitorias en el porcentaje o el número absoluto de linfocitos NK. Se caracterizaron, además, cinco pacientes con alteraciones persistentes en la distribución de las subpoblaciones de linfocitos NK. Estas alteraciones se debieron principalmente a la disminución de células CD56 dim CD16 bright . Se evidenciaron, también, defectos en la función de los linfocitos NK en algunos de nuestros pacientes; sin embargo, estas alteraciones fueron transitorias y se asociaron principalmente a la fase activa de la enfermedad. Conclusiones. Nuestros resultados evidencian defectos transitorios en el número y función de los linfocitos NK en pacientes con infecciones recurrentes o graves, además de alteraciones persistentes en los LNK CD56 dim CD16 bright en algunos individuos. Es necesario profundizar en los mecanismos que conllevan al desarrollo de estos defectos inmunes y estudiar cómo estas alteraciones influyen en la respuesta inmune.

Introduction: The information about defects affecting natural killer cell (NK) development and activity in patients with an abnormal increase of recurrent infections is scarce. Objective: To perform a systematic analysis of NK abnormalities in patients with recurrent infections. Materials and methods: Our study enrolled twenty patients with severe or recurrent viral infections. Natural killer cell subsets, surface receptors expression and cytotoxicity were analyzed. Results were compared with those from age- and sex-matched healthy controls. Results: Transient alterations were observed in the percentages and absolute numbers of NK cells in patients with infection active episodes. We also described five patients with stable disturbances in the distribution of NK cell subpopulations. These defects are mainly due to a decrease in the CD56 dim CD16 bright cells in peripheral blood. In addition, NK cell function abnormalities were observed in some patients, however, those were always transient and mainly associated to active disease. Conclusions: These findings demonstrate transient alterations in the percentages and absolute numbers of NK cells in patients with recurrent or severe infection. Also, stable disturbances in CD56 dim CD16 bright NK cells are observed in these patients. Nevertheless, these parameters must be thoroughly studied to determine the mechanisms that entail these immune abnormalities and investigate how they alter the immune response.

Preparation and cytotoxicity evaluation of chitosan-sodium alginate composite gel / 中国组织工程研究

BACKGROUND:Sodium alginate and chitosan are the polycation and polyanion natural polymer materials respectively, and they can be crosslinking agents complementing each other to form composite gel and avoid the cytotoxicity resulting from some common crosslinking agents . OBJECTIVE:To prepare the chitosan-sodium alginate composite gel and evaluate its cytotoxicity in vitro. METHODS:Chitosan was dissolved in 0.25 mol/L acetic acid to make a 30 g/L mass concentration solution, and 0.1 mol/L NaOH solution was added to neutralize its acidity. Neutralization of the chitosan solutions leads to the formation of a precipitate in ultrasmal particles. Then the chitosan and 3%sodium alginate solution in deionized water were mixed in 1:1 volume ratio by high frequency oscil ating to produce composite gel. The composite gel were detected by scanning electron microscopy and Fourier transform infrared spectrometry after freeze-drying. The 24-hour and 72-hour leaching solutions of composite gel, 24-hour and 72-hour leaching solutions of polyethylene and phenol solution were added to the L-929 cells’ culture medium respectively in order to evaluate the cytotoxicity of composite gel in vitro. RESULTS AND CONCLUSION:The results of Fourier transform infrared spectrometry showed the variation of characteristic peak values of composite gel which were different from sodium alginate and chitosan;and under scanning electron microscope, a spatial network structure formed with abundant intervals. Result of the cytotoxicity valuation was qualified for the chitosan-sodium alginate composite gel. These findings indicate that the chitosan-sodium alginate composite gel can be used as tissue engineering scaffold materials.

B-cell Complement Dependent Cytotoxic Crossmatch Positivity is an Independent Risk Factor for Long-term Renal Allograft Survival

The clinical significance of positive B-cell complement-dependent cytotoxicity crossmatching (B-CDC) in renal transplant recipients remains unclear. We reviewed 20 recipients with isolated B-CDC positivity at the time of transplantation. We compared the clinical characteristics, acute rejection and long-term graft survival between positive and negative B-CDC patients (n = 602). The number of retransplant recipients and positivity for T- and B-flowcytometric crossmatch was greater in positive B-CDC patients than in negative B-CDC patients. The overall acute rejection rate of positive B-CDC patients was significantly higher (P < 0.001), and Banff grade II or III cellular rejection was more frequently observed in positive B-CDC patients (P = 0.037). Compared with negative B-CDC patients, acute cellular rejection as a cause of graft loss was more prevalent (P = 0.020) and rescue rejection therapy was more frequently needed in positive B-CDC patients (P = 0.007). The allograft survival rate of positive B-CDC patients was significantly lower than that of negative B-CDC patients (P < 0.001), and B-CDC positivity independently increased the risk of allograft failure 2.31-fold (95% CI 1.15-4.67; P = 0.019) according to multivariate analysis. In conclusion, isolated B-CDC positivity is an independent long-term prognostic factor for allograft survival.

Expression and cytotoxic effect of transmembrane form of human blood group A antigen mimotope vaccine in a malignant melanoma cell line B16 / 中华皮肤科杂志

Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e.,P/F-M-pIRES group [transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein (Mip)-pIRES], P/F-pIRES group (transfected with the recombinant plasmid mimotope peptide/FaspIRES), M-pIRES group (transfected with the recombinant plasmid Mip-pIRES), and pIRES group (transfected with the empty plasmid pIRES). B 16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 (CCK-8) was used to evaluate the vaccinemediated complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC (F = 244.522, P ＜ 0.01 ) and ADCC (F = 71.593, P ＜ 0.01 )against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/FM-pIRES and P/F-pIRES groups compared with M-plRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group (F = 15.42, P ＜ 0.05), but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.

Basic sieving of an injectable prosthetic nucleus pulposus by studying cytotoxicity in vitro / 中华骨科杂志

To sieve a new type of polyether polyurethane as an injectable prosthetic nucleus pulpous through testing the cytotoxicity in vitro.Methods Five experimental groups were set as following:that samples 1,2,3 groups were polyether-polyurethane polymerized of different ratio of soft and hard segment with different catalysts; group 4 was polyether polyurethane polymerized slowly at 37 ℃ without catalyst; group 5was polyether polyurethane that toxic monomer responded completely.Additionally,a control group of culture medium was set without material extracts.Get 0.6 mg from each sample product and make into material extracts after disinfection,then dilution them by 50% and culture the mouse fibroblasts(cell L929)until 3 d,4d,5 d,by Adopting 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetric method to compare the 490 nm OD between the materials and control groups obtained by enzyme linked immune analysis,calculate the RGR of cells,and then evaluating the cytotoxicity of the materials.Results The RGR of L929 cell in group 4 and group 5 have no significant difference compared to the control group.Cytotoxic levels were 0 to 1; difference of the RGR of L929 cell between group 1-3 and control group were statistically significant.Cytotoxic levels were 1 to 2,but the cell morphology was normal.Conclusion The material polymerized without catalyst does not show cytotoxicity,and has great potential to be used as a substitute for nucleus pulposus.The cytotoxicity of the materials polymerized with catalyst needs to be tested more.

Cytotoxicity of current adhesive systems: in vitro testing on cell culture of L929 and balb/c 3T3 fibroblasts / Citotoxicidade de sistemas adesivos atuais: teste in vitro em cultura de células fibroblásticas L929 e balb/c 3T3

Purpose: The aim of this study was to evaluate the cytotoxicity of three current adhesives: Prime&Bond NT (PBNT), Single Bond (SB) and XENO III (XENO). Methods: After embedding and curing circles of filter paper with the tested adhesives, the filters were placed in contact with the solidified agar surface over L929 monolayer cells plated in 6-well cell culture plate and incubated for 24 h. The inhibition zone around the filter papers was measured in mm. MTT assay was performed using fibroblasts Balb/c 3T3 cell lines in multiwell culture plates. All assays were done in triplicate. Results: All materials were cytotoxic (Kruskal-Wallis, P<0.05) in a similar level to latex (P>0.05). For intra-groups analysis, SB presented the lowest cytotoxicity (P<0.01), while there was no statistical difference between PBNT and XENO (P>0.05). MTT assay confirmed the cytotoxicity of the tested adhesives. Conclusion: Considering the limits of this work, all adhesives tested were as cytotoxic as latex.

Objetivo: O objetivo deste estudo foi avaliar a citotoxicidade de três adesivos: Prime & Bond NT (PBNT), Single Bond (SB) e XENO III (XENO). Metodologia: Após embebição e polimerização de filtros de papel com os referidos adesivos, estes foram colocados em contato com a superfície de agar solidificada sobre a monocamada de células L929 plaqueadas em cultura celular de 6-poços e incubadas por 24 h. A zona de inibição formada ao redor dos filtros de papel foi medida em milímetros. Outro teste realizado foi o do MTT, utilizando fibroblastos Balb / c 3T3 em placas de multi-poços, sendo os ensaios realizados em triplicatas. Resultados: Todos os materiais testados foram citotóxicos (Kruskal-Wallis, P < 0,05) e semelhantes ao látex (P > 0,05). Para a análise intra-grupos, o SB apresentou a mais baixa citotoxicidade (P < 0,01), enquanto não houve diferença estatística entre PBNT e XENO (P > 0,05). O ensaio de MTT confirmou a citotoxicidade dos adesivos. Conclusão: Considerando as limitações deste trabalho, todos os adesivos testados foram tão citotóxicos quanto o látex.

Cytotoxicity of polycarbonate orthodontic brackets

Aim: To assess the cytotoxicity of polycarbonate orthodontic brackets. Methods: Polycarbonate brackets from two different manufacturers, namely, Composite bracket (Morelli™) and Silkon Plus bracket (American Orthodontics™), were assessed. In addition to these two experimental groups, other three control groups were included: Positive Control Group (C+) consisting of amalgam cylinders, Negative Control Group (C-) consisting of glass rods, and Cell Control Group (CC) consisting of cells not exposed to any material. All brackets were previously sterilized under ultra-violet light (UV) and, then, immersed in Eagle’s minimum essential media (MEM) for 24 hours, after which the supernatants were removed and placed into contact with L929 fibroblast cells. Cytotoxicity was evaluated at 24, 48, 72 and 168 hours. After contact with MEM, the cells were further incubated at 37oC for 24 hours and 100 mL of 0.01% neutral red dye were added. The cells were incubated again at 37ºC for three hours to incorporate the dye. After this period, the cells were fixed and viable cell counting was performed by spectrophotometry at 492 nm wavelength. Results: No statistically significant difference was found between the experimental groups (1 and 2) and the negative and cell control groups (p > 0.05). The Positive Control Group exhibited high cytotoxicity throughout experimental period are differed significantly from the other groups (p < 0.05). Conclusions: Polycarbonate orthodontic brackets were found not to be cytotoxic within the evaluated experimental period.

Expression of membrane inhibitor of reactive lysis in non-small cell lung carcinoma and its clinical significance / 中国基层医药

Objective To survey membrane inhibitor of reactive lysis(MIRL) expression in non-small cell lung careinoma(NSCLC) and to analyze the relationship between MIRL expression and clinical staging, adjuvant chemotherapy and disease-free survial. Methods The expression of MIRL in 8 adjacent tissues and 36 NSCLC sam-pies were determined by immunohistochemistry. Furthermore, the relationship between MIRL expression and clinical stage ,adjuvant chemotherapy and disease-free survival was assayed by follow-up. Results Among 36 samples of non-small-cell lung cancer,there were 10(27.8%) samples expressing MIRE. Out of 18 samples of squamous carcinoma, 4(22.2%) expressed MIRL,while 6(37.5%) expressed it in 16 samples of adenocarcinoma,there was no statistical significance between them(P>0.05). There were no expression in 2 samples of large cell carcinoma. There was no correlation between MIRL expression and disease-free survival(P>0.05). MIRL positive expression rate in patients with preoperational adjuvant chemotherapy was significantly lower than that of those without preoperational adjuvant chemotherapy(P<0.05). Conclusions There is great percentage of MIRE expression in NSCLC. Our present study suggests that the immunological inhibition of MIRL should be blocked when monoclonal antibody is used in the treat-merit of NSCLC.