The influence of demography and referral medical specialty on the detection of autoantibodies to HEP-2 cells in a large sample of patients

Abstract Background: The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. Methods: A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. Results: From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively ( p < 0.0001). The proportion of positive results in the 4 age groups analyzed: 0-19, 20-39, 40-59, and ≥ 60 years was 23.3, 20.2, 20.1, and 22.8%, respectively ( p < 0.0001). Considering all positive sera (n = 11,478), AC-4 nuclear fine speckled (37.7%), AC-2 nuclear dense fine speckled (21.3%), BAC-3 nuclear quasi -homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) ( p < 0.0001). Moderate (46.4%) and high (10.8%) titers were more observed in patients referred by rheumatologists ( p < 0.0001). We observed a high proportion of mixed and cytoplasmic patterns in samples referred by oncologists and a high proportion of BAC-3 (nuclear quasi -homogeneous) pattern in samples referred by pneumologists. Conclusions: One-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0-19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoanti-bodies were more frequent in patients referred by rheumatologists.

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma / Diagnóstico e tratamento de células HEp-2 contaminadas por micoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

Effect of miR-340-5p on proliferation of laryngeal cancer Hep2 cells and its intrinsic molecular mechanism / 临床耳鼻咽喉头颈外科杂志

To study the effect of miR-340-5p on the proliferation of laryngeal cancer Hep2 cells and explore its intrinsic molecular mechanism, so as to screen potential biomarkers and targets for the diagnosis and treatment of laryngeal cancer. The expression of miR-340-5p in laryngeal cancer tissues, paracancerous tissues, laryngeal cancer cell lines Hep2 and normal bronchial HBE cell lines was quantitatively analyzed by qRT-PCR; The double luciferase reporter vector was constructed to verify whether STAT3 was a potential target gene of microRNA-340-5p; The miR-340-5p mimics/inhibitor was transfected into Hep2 cells by liposome and verified by qRT-PCR; The CCK-8 method and Annexin V/PI method were used to analyze the proliferation and apoptosis of transfected cells; and Western Blot was used to detect the expression of STAT3 and Wnt/β-catenin pathway-related proteins after transfection. The results of qRT-PCR showed that the level of miR-340-5p in laryngeal cancer tissues and Hep2 cells was significantly lower than that in adjacent tissues and HBE cells, and the expression of miR-340-5p was significantly increased or decreased after overexpression or inhibition; Luciferase activity showed that miR-340-5p directly interacted with target gene STAT3 3'-UTR and negatively regulated its expression; Cell proliferation and apoptosis analysis showed that up-regulation of microRNA-340-5p could significantly inhibit the proliferation and induce apoptosis of Hep2 cells in vitro, and vice versa; Western Blot results showed that the levels of STAT3 and β-catenin, c-Myc, TCF-4, CyclinD1 and ROCK1 in Hep2 cells were significantly lower than those in the control group after over-expression of miR-340-5p, and vice versa. The expression of miR-340-5p is abnormally low in laryngeal cancer tissues and Hep2 cells. It can be used as a potential biological target for diagnosis and treatment of laryngeal cancer by targeting STAT3 gene to negatively regulate Wnt/β-catenin signaling pathway.

Inhibitory effect of miR-3195 on the proliferation of human laryngeal carcinoma Hep-2 cells and its mechanism / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of miR-3195 on the proliferation of laryngeal carcinoma Hep-2 cells and its molecular mechanism. Methods: From January 2008 to August 2012, the laryngeal cancer tissues and their corresponding paracancerous tissues from 29 patients with laryngeal cancer who were admitted to the Department of Otorhinolaryngology, Chenzhou First People's Hospital Affiliated to teaching hospital of University of South China were selected for this study. qPCR was used to detect the expression of miR-3195 in laryngeal carcinoma and the paracancerous tissues; Hep-2 cell line with stable and high expression of miR-3195 was constructed. The proliferation of miR-3195 over-expressed Hep-2 cells and the control cells was observed by MTT method. A nude mouse xenograft model was established to observe the proliferation of miR-3195 overexpressed Hep-2 cells in nude mice. Bioinformatics tools were used to predict the target gene of miR-3195; the luciferase vector of TBX1 3'UTR was constructed, and its luciferase activity was examined with dual luciferase detection system; Western blotting was used to detect the TBX1 protein expression in miR-3195 over-expressed cells and control cells. Results: The expression of miR-3195 in laryngeal carcinoma tissues was significantly lower than that in paracancerous tissues (P<0.01); miR-3195 up-regulation could inhibit the proliferation of Hep-2 cells (P<0.01) and significantly inhibit the growth of transplanted tumors in nude mice (P<0.05); The results of the Dual luciferase reporter gene assay indicated that miR-3195 might targetedly bind to TBX1 (P<0.05), and Western blotting proved that miR-3195 could inhibit the expression of TBX1 protein (P<0.05). Conclusion: miR-3195 has a significant inhibitory effect on the proliferation of Hep-2 cells, and its molecular mechanism may be related to the negative regulation of TBX1 expression.

Effect of miR-340-5p on proliferation of laryngeal cancer Hep2 cells and its intrinsic molecular mechanism / 临床耳鼻咽喉头颈外科杂志

Objective@#To study the effect of miR-340-5p on the proliferation of laryngeal cancer Hep2 cells and explore its intrinsic molecular mechanism, so as to screen potential biomarkers and targets for the diagnosis and treatment of laryngeal cancer. @*Method@#The expression of miR-340-5p in laryngeal cancer tissues, paracancerous tissues, laryngeal cancer cell lines Hep2 and normal bronchial HBE cell lines was quantitatively analyzed by qRT-PCR; The double luciferase reporter vector was constructed to verify whether STAT3 was a potential target gene of microRNA-340-5p; The miR-340-5p mimics/inhibitor was transfected into Hep2 cells by liposome and verified by qRT-PCR; The CCK-8 method and Annexin V/PI method were used to analyze the proliferation and apoptosis of transfected cells; and Western Blot was used to detect the expression of STAT3 and Wnt/β-catenin pathway-related proteins after transfection. @*Result@#The results of qRT-PCR showed that the level of miR-340-5p in laryngeal cancer tissues and Hep2 cells was significantly lower than that in adjacent tissues and HBE cells, and the expression of miR-340-5p was significantly increased or decreased after overexpression or inhibition; Luciferase activity showed that miR-340-5p directly interacted with target gene STAT3 3'-UTR and negatively regulated its expression; Cell proliferation and apoptosis analysis showed that up-regulation of microRNA-340-5p could significantly inhibit the proliferation and induce apoptosis of Hep2 cells in vitro, and vice versa; Western Blot results showed that the levels of STAT3 and β-catenin, c-Myc, TCF-4, CyclinD1 and ROCK1 in Hep2 cells were significantly lower than those in the control group after over-expression of miR-340-5p, and vice versa. @*Conclusion@#The expression of miR-340-5p is abnormally low in laryngeal cancer tissues and Hep2 cells. It can be used as a potential biological target for diagnosis and treatment of laryngeal cancer by targeting STAT3 gene to negatively regulate Wnt/β-catenin signaling pathway.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

Abstract Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

Effect of MiR-1264 on the proliferation and migration of Hep2 cell line / 医学研究生学报

Objective Previous study shows that the expression of mir-1264 is down-regulated in laryngeal carcinoma. This study aimed to investigate the effect of miR-1264 on the biological function of laryngeal carcinoma Hep2 cells and verify whether miR-1264 could down-regulate the expression of tumor suppressor gene la-ryngeal carcinoma related gene 1(LCRG1)in laryngeal carcinoma. Methods Real-time quantitative PCR was used to assess the miR-1264 expression in human laryngeal cancer tissues. There are three groups in the experiment:miR-1264 mimic/inhibitor group,mimic/inhibitor NC group and untransfected group. MTT and Transwell were applied to observe the effect of miR-1264 on the proliferation,migration and invasion capabilities of Hep2 cells. Luciferase experiment was used to verify the combination of miR-1264 and LCRG1 3'UTR.RT-PCR and Western blot were used to test the expression of miR-1264 and LCRG1 protein respectively. Results Compared with the adjacent laryngeal tissue,miR-1264 expression in human laryn-geal cancer tissues was significantly increased(P<0.05).Compared with NC control group and blank group,the proliferation,migration and invasion capabilities of Hep2 cells were significantly enhanced after they were transiently transfected with miR-1264 mimic(P<0.05);however,after Hep2 cells were transiently transfected with miR-1264 inhibitor,their proliferation,migration and invasion ca-pabilities were significantly inhibited(P<0.05). Luciferase experiment showed miR-1264 mimic could significantly decrease LCRG1 3'UTR luciferase activity,which was of statistical significance. After the verification of successful transient transfection,the results of further Western blot on LCRG1 protein expression showed that there was no significant difference between the experimental group and the mimic NC control group as well as the blank group(P>0.05). Conclusion miR-1264 is highly expressed in laryngeal cancer tissues. Though miR-1264 may not participate in down-regulating the expression of tumor suppressor gene LCRG1 in laryngeal carcino-ma,it can promote the proliferation,migration and invasion capabilities of Hep2 cells.

Impact of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep2 / 中国免疫学杂志

Objective:To study the effect of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep-2.Meth-ods:The oligomucleotide of miR-122a was transfected into laryngeal carcinoma cell line Hep 2 cells,which were devided into three groups of A(miR-122a transfection),B(miR-122a inhibitor),C(miR-122a-NC inhibitor) and group of D(blank control).The expres-sion of miR-122a was defected by RT-PCR,and relevant protein expression was evaluated by Western blot .The cell proliferation and cell cycle were determined by MTT assy and flow cytometry ,respectively.Results:Compared to group D,miR-122a expression in Hep2 cells was obviously elevated atter miR-122a-transfected.The proliferation of Hep2 cells in group A was significantly inhibited and the cell cycle arrested at G1/G0 phase.The protein expression of CDC42 was downregulated with decreased expressions of CDK 4 and cyclin D1 in group A.Conclusion:miR-122a inhibits the proliferation activity of Hep 2 cells,suggesting that miR-122a can be taken as a po-tential candidate for gene therapy of laryngeal carcinoma .

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

The research on apoptosis of human laryngeal cell line Hep-2 induced by 1,4-bis[2-(benzylselanyl) ethoxy] anthracene / 中国癌症杂志

Background and purpose:Selenium is one of the essential trace elements for human activities, and plays an incomparable role in maintaining human health. It was reported that selenium compound 1,4-bis[2-(benzylse-lanyl)ethoxy] (BSEA) anthracene has antiseptic and antiphlogistic effects. However, the mechanisms underlying anti-cancer effects of BSEA are rarely reported. BSEA-induced apoptosis in human laryngeal carcinoma Hep-2 cells and its mechanisms were studied.Methods:Methyl thiazolyl tetrazolium (MTT) assay was used to determine inhibition ratio of Hep-2 cells 24 hours after Hep-2 cells were treated with different concentrations of BSEA. Fluorescence microscope was used to observe the morphology change of apoptosis in Hep-2 cells. The apoptosis was detected by Annexin Ⅴ-FITC. Mi-tochondrial membrane potential was assayed by JC-1. Microplate reader detected the activity of caspase-3 and caspase-8. The mRNA and protein levels of Bax and XIAP were measured by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot.Results:The results showed that BSEA caused a dose-dependent inhibition of the growth of human laryngeal carcinoma cell line Hep-2in vitro, andIC50 was 35.74μmol/L. The apoptotic bodies were distinctly observed at a concentration of 80μmol/L of BSEA by AO fluorescence staining. This study found that the eversion of phosphatidyl serine intensified, and mitochondrial membrane potential also began to decline. The activity of caspase-3 appeared the tendency of dependence on dosage, while the activity of caspase-8 did not change significantly. The mRNA and protein expression level of Bax increased, whereas the mRNA and protein expression level of XIAP de-creased.Conclusion:Therefore, BSEA could obviously inhibit human laryngeal carcinoma Hep-2 cells proliferation and induce apoptosis via the mitochondrial pathway.

Fluid shear stress regulates epithelial-mesenchymal transition (EMT) in Hep2 cells / 医用生物力学

Objective To examine the effects of fluid shear stress (FSS) on epithelial-mesenchymal transition (EMT) in Hep2 cells. Methods Hep2 cells were exposed to 140 mPa FSS. The morphologic changes of Hep2 cells exposed to FSS at different durations were observed using inverted microscope. The migration ability of Hep2 cells after FSS loading was investigated using scratch wound assay. The distribution and expression of cytoskeleton protein F-actin were examined by confocal microscope. The expression of the EMT marker proteins were detected by Western blotting. Results Most of Hep2 cells changed their morphology from polygon to elongated spindle with well-organized F-actin under FSS. After removing FSS, Hep2 cells recovered their initial morphology with flat polygon. FSS regulated Hep2 cells to enhance their migration capacity in a time-dependent manner. FSS promoted the rearrangement of cytoskeletal protein F-actin，which enhanced the migration behavior of Hep2 cells. In addition, FSS induced a time regularity of expression of the EMT marker proteins in Hep2 cells. Conclusions FSS as an important physical factor can induce EMT in Hep2 cells.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

OBJECTIVE@#To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells.@*METHODS@#The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells.@*RESULTS@#Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P < 0.01), the cell apoptosis rate was significantly increased (P < 0.01), the abilities of cell migration and invasion were significantly reduced (P < 0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P < 0.01).@*CONCLUSIONS@#The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

Objective To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. Methods The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells. Results Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P < 0.01), the cell apoptosis rate was significantly increased (P < 0.01), the abilities of cell migration and invasion were significantly reduced (P < 0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P < 0.01). Conclusions The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegansand the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.

Comparación del cultivo celular de HeLa y HEp-2: Perspectivas de estudios con Chlamydia trachomatis / Comparison of cell culture HeLa and HEp-2: Studies prospects Chlamydia trachomatis

Objetivo. Estandarizar el cultivo de células HeLa en diferentes condiciones, con el fin de utilizarlo en protocolos de infección con Chlamydia trachomatis serovar L2. Materiales y métodos. Este estudio se llevó a cabo en cuatro fases principales que son: 1.Viabilidad celular por la técnica de azul de tripán y su posterior observación, 2. Estandarización del cultivo de células HeLa, 3. Coloración de Giemsa, 4. Cultivo de células HEp-2.Resultados. Se determinó que la línea celular HeLa debe ser cultivada en medio DMEM, 0,1% de L- glutamina, 10% de SFB. Así mismo, que la coloración de Giemsa es mejor realizarla en un tiempo de 40 minutos por que se evidencia una clara definición de núcleo y citoplasma. Frente a la comparación de las dos líneas celulares se obtuvo que la línea HeLa desde el primer día muestra un crecimiento adecuado y alcanza rápidamente la confluencia esperada, en contraposición la línea HEp-2 presenta un crecimiento más lento pero alcanzando la confluencia deseada al último día.

Objective. The goal of this study was to standardize the cultivation of HeLa cells under different conditions, to be used in Chlamydia trachomatis serovar L2 infection's protocols. Materials and Methods. This study was conducted in four phases that are: 1.Cell Viabilidad by trypan blue technique and his subsequent remark, 2. Standardization HeLa cell culture, 3. Giemsa, 4. Cultivation of Hep-2 cells. Results. As a result of standardization it is determined that the HeLa cell line should be cultured in DMEM, 0.1% L-glutamine, 10% FBS.It was determined that the Giemsa perform better over time of 40 minutes that a clear definition of nucleus and cytoplasm is evident. Comparing against both cell lines HeLa was obtained that the line from day growing well and quickly reaches the expected confluence, the opposed line HEp-2 has a slower growth but achieving the desired confluence the last day.

Pharmacodynamic Study on in vitro Antiviral Effect of Junduqing Granules / 中国药房

OBJECTIVE:To study in vitro antiviral effect of Junduqing granules. METHODS:Human laryngeal cancer Hep2 cells were inoculated with the Nancy strain of coxsackievirus B3 (CoxB3) and respiratory syncytial virus (RSV) to establish vi-rus-infected cell models. 2 mg/ml original liquid of Junduqing granules and 2 mg/ml original liquid of chlorogenic acid which were diluted according to the ratio of 1∶2-1∶256 were used to act on the virus-infected cells. Reed-Muench method was adopted to calcu-late 50% toxic concentration (TC50) and maximal atoxic concentration (TC0). Virus-infected cells were cultured with solution of Junduqing granules of 1.25,0.625,0.312 5 and 0.156 25 mg/ml and chlorogenic acid solution 0.5,0.25,0.125,0.062 5 mg/ml, and then the degree of cytopathic effect was evaluated with a microscope. Virus-infected cells were cultured with 1.25,0.625, 0.312 5 ,0.156 25,0.078 125 mg/ml solution of Junduqing granules and 0.5,0.25,0.125,0.062 5,0.031 25 mg/ml of chlorogenic acid solution,and Reed-Muench method was adopted to calculate 50% inhibitory concentration (IC50) and therapeutic index (TI) on RSV and CoxB3 cells. RESULTS:TC50 of Junduqing granules was 1.79 mg/ml and TC0 was 1.25 mg/ml,TC50 of chlorogenic ac-id was 0.71 mg/ml and TC0 was 0.5 mg/ml. Virus-infected cells grew normally when the mass concentration of solution of Jundu-qing granules was 1.25 mg/ml. IC50 of Junduqing granules was 0.22 mg/ml and TI was 8.14;IC50 of chlorogenic acid were 0.18 and 0.36 mg/ml,TI were 3.94 and 1.97. CONCLUSIONS:Junduqing granules have in vitro anti-CoxB3 and RSV effect.

Effects of phycocyanin on apoptosis of human laryngeal cancer HEP-2 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of phycocyanin on the apoptosis of human laryngeal cancer HEP-2 cells and to explore the inhibitory mechanism of phycocyanin to tumor.METHODS:Highly purified phycocyanin was ex-tracted from spirulina.The effects of phycocyanin at different concentrations on the growth of human laryngeal cancer HEP-2 cells were detected by MTT assay.In addition, the cell structures were observed under electron microscope.The cell ap-optosis was analyzed by flow cytometry.The production of reactive oxygen species ( ROS) was measured by flow cytometry. Enzymatic activities of caspase-3,-8 and-9 were measured by chemical colorimatry.The expression of Bax, Bcl-2, Fas, P53, caspase-3 and caspase-9 at mRNA and protein levels was determined by RT-PCR and Western blot.RESULTS:MTT test confirmed that phycocyanin inhibited the cell activity of HEP-2 cells with time and dose dependent manners.The result of electron microscope observation and flow cytometry indicated that phycocyanin induced the apoptosis of HEP-2 cells.The intracellular content of ROS was increased.The activities of caspase-3, -8 and -9 were increased.RT-PCR showed that the mRNA expression of Bax, Fas, P53, caspase-3, caspase-9 was increased and Bcl-2 was decreased.The results of Western blot were consistent with the results of RT-PCR.CONCLUSION:Phycocyanin might induce apoptosis of HEP-2 cells by down-regulating Bcl-2, up-regulating Bax, Fas and P53, and the transduction of apoptotic signals in the human laryngeal cancer cells.

IV Consenso Brasileiro para pesquisa de autoanticorpos em celulas HEp-2 / IV Brazilian Guidelines for autoantibodies on HEp-2 cells

Objetivo: O IV Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) realizado em Vitória (ES), no dia 18 de setembro de 2012, objetivou discutir estratégias e recomendações relacionadas ao procedimento técnico, à padronização e à interpretação dos resultados da pesquisa de autoanticorpos em células HEp-2. Métodos: Participaram do evento 23 pesquisadores e especialistas de Universidades e laboratórios brasileiros. Foram abordados diferentes tópicos, discutidos amplamente a fim de se estabelecer recomendações específicas. Resultados e conclusão: O IV Consenso integrou à árvore de decisão o padrão citoplasmático em Anéis e Bastões, o padrão nuclear pontilhado Quasi-homogêneo (QH) e o padrão misto CENP-F. Discutiu-se ainda a necessidade de atenção para a classificação do padrão misto relacionado à presença de anticorpos anti-DNA topoisomerase I (Scl70), compreendendo os componentes nuclear pontilhado fino, nucleolar homogêneo, NOR na placa metafásica e citoplasmático pontilhado fino. Foram sugeridas diretrizes para o controle de qualidade do teste, diluição de triagem e diluição de esgotamento, e foi emitido alerta quanto à necessidade de atenção em relação à heterogeneidade de substratos disponíveis no mercado e a utilização de metodologias automatizadas para detecção de autoanticorpos. .

Objective: The Fourth Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) was held in Vitória, Espírito Santo, and aimed to discuss strategies and recommendations about the technique, standardization, interpretation and quality control of the indirect immunofluorescence reaction on HEp-2 cells. Methods: Twenty three ANA experts from university centers and private laboratories in different areas from Brazil discussed and agreed upon recommendations for the fourth edition of the Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells. Results and conclusion: The 4th ANA Consensus included three novel patterns into the existing algorithm (cytoplasmic Rods and Rings, nuclear Quasi-homogeneous, and CENP-F). Emphasis was given to the need of attention in describing the peculiar mixed pattern elicited by anti-DNA topoisomerase I (Scl-70) autoantibodies, comprising nuclear fine specked, nucleolar homogeneous pattern, NOR staining in metaphase plates, and cytoplasmic fine speckled patterns. The group also emphasized the need for continuous quality control in indirect immunofluorescence assays, the establishment of screening dilutions, as well as conjugate titration. An alert was made regarding the heterogeneity of commercial kits in defining patterns and the use of solid phase methodologies to determine the presence of autoantibodies. .

α-Lipoic acid promotes inhibition of pyrroIidine dithiocarbamic acid on Hep-2 ceII proIiferation / 中国药理学与毒理学杂志

OBJECTIVE To investigate whether α-lipoic acid(α-LA)can promote the anti-prolifera-tion effect of pyrrolidine dithiocarbamic acid(PDTC)in Hep-2 cells. METHODS The laryngeal carcino-ma Hep-2 cells were cultured with α-LA,PDTC or α-LA+PDTC respectively for 12,24 and 48 h. The proliferation of Hep-2 cells was detected by WST-1 assay and soft agar colony formation while apoptosis and cell cycle were analyzed by Hoechst33258 staining and flow cytometry. RESULTS α-LA 5 -500 μmol·L-1 could not inhibit Hep-2 cell proliferation,but PDTC 5 -50 μmol·L-1 could( P ﹤0.05,P ﹤0.01). The cell proliferation inhibitory rate of PDTC 5 μmol·L-1 combined with α-LA 5,10 and 20 μmol·L-1 groups was much higher than in control group(P﹤0.01)and PDTC alone group(P﹤0.05). When α-LA 5 μmol·L-1 was used in combination with PDTC 5 μmol·L-1 for 12-48 h,the cell proliferation was inhibited in a time-dependent manner(r=0.987,P﹤0.05). When the cells were treated for 24 h,the number of soft agar colony formations in combined group was significantly smaller than that of both α-LA alone group(P﹤0.01)and PDTC alone group(P﹤0.05). The result of Hoechst33258 staining and flow cytometry indicated that after combined treatment with PDTC 5 μmol·L-1 and α-LA 5 μmol·L-1 for 24 h,the level of apoptosis and the percentage of cells in G2 / M stage were significantly increased compared with PDTC alone or α-LA alone treatment. CONCLUSION α-LA can enhance the anti-proliferation and pro-apoptosis effect of PDTC in Hep-2 cells.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location ofUreaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversuminvasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.