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Six new derivatives were synthesized by reacting the alkaloid songorine (1) with isocyanates. Their structures were confirmed by IR, mass, 1H and 13C NMR spectral methods. The effects of the obtained compounds on tumor cell growth in vitro were studied. 21-Ethyl-1-O-3′-chlorophenylcarbamate-15-hydroxy-4-methyl-16-methylene-7,20-cycloveatchan-12-one (3c) exhibited cytotoxicity against HeLa and HEp-2 lines at a concentration of 100 μg/mL. The other songorine derivatives caused dose-dependent proliferation of cancer cells. [ABSTRACT FROM AUTHOR]

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Two new series of 4,5-difunctionalized 1-bromobenzyl[1,2,3]triazole (2a, 3a, 4a, 5a, and 6a) and 4,5-difunctionalized 1-(2-oxo-2-(p-tolylamino)ethyl-[1,2,3]triazole (2b, 3b, 4b, 5b, and 6b) were synthesized using related 1-(azidomethyl)-4-bromobenzene 1a and p-tolylcarbamoyl azide 1b respectively. The substituted [1,2,3]triazolo[4,5-d]pyrimidine-7-one derivatives (7a, 7b, 8a, and 8b) were synthesized by the reaction of [1,2,3]triazolo derivatives 2a and 2b with carbon disulfide in the presence of 10% sodium hydroxide/dimethylformamide and/or by the reaction with formic acid respectively. The S-glucoside derivatives (9–12) of newly synthesized [1,2,3]triazolo[4,5-d]pyrimidines were also synthesized. By using several spectroscopic methods, including IR, 1H NMR, 13C NMR, and elemental analysis, the chemical structures of the novel derivatives were confirmed. The synthetic compounds' cytotoxicity and in vitro anticancer activity were examined vs. human breast carcinoma (MCF-7), human laryngeal carcinoma (HEP-2), and human colorectal carcinoma (HCT-116) cell lines. According to the findings, HEP-2 and HCT-116 cells are more sensitive to the tested compounds than the other cell lines. In the HEP-2 and MCF-7 cell lines, respectively, compounds 5b and 11 showed potential anticancer activity when compared to the effect of the commonly used anticancer medication, doxorubicin. The selectivity of compounds against the cancer cell line was confirmed by testing their cytotoxicity on VERO (African Green Monkey kidney) normal cells. The anticancer activity of those compounds is suggested to be due to nuclear damage generated by a high generation of reactive oxygen species (ROS). In addition, the induction of apoptosis by significantly upregulating the apoptotic genes PAR-4 and BAX while substantially downregulating the anti-apoptotic genes BCL-2 and BCL-xl. Molecular docking research was undertaken to predict the probable binding poses of the most effective drugs in the active site of CDK-2. The more active compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) have been docked on the CDK-2 enzyme to demonstrate their mode of action as anticancer medicines. The compounds exert many interactions and showed high binding to the CDK-2 receptor. Finally, a hypothetical pharmacophore model was created using the Molecular Operating Environment (MOE) software and five compounds that are structurally similar to the synthesized ones with known anticancer action. [ABSTRACT FROM AUTHOR]

Objective: The HEp-2 cell line was first identified as laryngeal cancer cells. Then, it was reported to consist of cervical adenocarcinoma cells derived via HeLa cell line contamination. Styrax liquidus is an exudate that is provided by the injured hull of the Liquidambar orientalis Miller (Hamamelidaceae), which has been used for the treatment of skin problems, peptic ulcers, and parasitic infections or as an antiseptic. In our study, we purposed to research the cytotoxic and apoptotic effect of styrax liquidus on HEp-2 cancer cell line. Materials and Methods: The IC50 dosage of styrax liquidus (Turkish sweet gum obtained from trees) was set by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the apoptotic effect of styrax liquidus on HEp-2 cancer cell was determined by assessing the expression of genes involved in apoptosis (Bax, Bad, Bak1, p53, Bcl-2, Bcl-XL, Apaf-1, Caspase2, Caspase3a, Caspase9, and Caspase12) by quantitative real-time polymerase chain reaction. Results: The IC50 value of styrax liquidus was found to be 125 µg/mL for 48 hours. According to the results, styrax liquidus reduced the population of HEp-2 laryngeal cancer cells and increased the expression of genes which were apoptosis related. These results indicate that styrax liquidus can be thought as a choice of cancer therapy. Conclusion: The finding of the study showed that it would be more useful to perform more qualified studies about the effect of styrax liquidus on cancer cells. [ABSTRACT FROM AUTHOR]

The resolution of complex medical diagnoses using pattern recognition requires an artificial neural network-based expert system to automate autoimmune disease diagnosis in blood samples. This process is done using image-based computer-aided diagnosis (CAD) to reduce errors in the diagnosis process. This paper describes a Multistage Classification Scheme (MSCS), which uses antinuclear antibody (ANA) tests to identify and classify the existence of autoantibodies in the blood serum that bind to antigens found in the nuclei of mammalian cells. The MSCS classified HEp-2 cells into three stages by using Binary Tree (BT), Artificial Neural Network (ANN), and Support Vector Machine (SVM) as basic blocks. The Indirect Immunofluorescence (IIF) technique is used in the ANA test with Human Epithelial type-2 (HEp-2) cells as substrates. The efficiency of the proposed methodology is assessed using the dataset of ICPR 2016. The intermediate cells (IMC) and positive cells (PC) were separated in Stage 1 prior to preprocessing based on their total strength, and special preprocessing is applied to intermediate cells for improved output, and positive cells are subjected to mild preprocessing. The mean class accuracy (MCA) was 84.9% for intermediate cells and 95.8% for positive cells, although the carefully picked 24 features and SVM classifier were applied. ANN showed better performance by adjusting the weights using the SCGBP algorithm. So, the MCA is 88.4% and 97.1% for intermediate and positive cells, respectively. BT had an MCA of 95.3% for intermediate and 98.6% for positive. In Stage 2, the meta learners BT2, ANN2, and SVM2 were trained for an augmented feature set (24 + 3 results from base learners). Therefore, the performance of BT2, ANN2, and SV M2 was increased by 1.8%, 4.5%, and 4.1% as compared to Stage 1. In Stage 3, the final prediction was performed by majority voting among the results of the three meta learners to achieve 99.1% MCA. The proposed algorithm can be embedded into a CAD framework built for the ANA examination. The proposed model will improve operational efficiency, decrease medical expenses, expand accessibility to healthcare, and improve patient safety in the sector, enabling enterprises to lower unplanned downtime, develop new products or services, increase operational effectiveness, and enhance risk management. [ABSTRACT FROM AUTHOR]

Abstract Objective To evaluate the perception of rheumatologists regarding the recommendations of the Brazilian Consensus for detection of Autoantibodies (BCA) on HEp-2 Cells by Indirect Immunofluorescence assay (IFA) and how BCA recommendations help in clinical practice. Methodology A structured questionnaire regarding the BCA recommendations for detection and interpretations of autoantibodies in HEp-2 cells was applied to randomly selected rheumatologists. The results were tabulated using the Microsoft® Excel program, expressed as a simple percentage and the dichotomous data were analyzed using the Chi-square test and the Epi Info® program. Results Four hundred fuorteen rheumatologists participated in the study: 70% of them considered their knowledge of the HEp-2 IFA test satisfactory or excellent, and 43% said they knew the BCA recommendations in general, without distinguishing the edition of the BCA to which they refer. The Revista Brasileira de Rheumatologia/Advances in Rheumatology was the means of dissemination most consulted by specialists (50%). According to the rheumatologists’ opinion, the most relevant pattern was the homogeneous nuclear (78%) and 65% stated they were satisfied with the BCA recommendations at a level of satisfaction greater than or equal to 80%. There was no significant difference in the perception of rheumatologists from the several Brazilian geographic regions. Conclusion Brazilian rheumatologists are aware of the BCA guidelines and most are satisfied with the content published, considering that the BCA recommendations assist positively in the clinical practice. Most rheumatologists recognize the patterns associated with rheumatic autoimmune diseases and have used BCA recommendations to interpret the results of the HEp-2 IFA test.

Routine cell culture demands the use of animal-derived products, mainly fetal bovine serum and swine or bovine trypsin. According to the 3Rs principle and to the European Centre for the Validation of Alternative Methods, animal-free substitutes are strongly recommended for in vitro methods. In this study, the HEp-2 cell line was adapted to different totally animal-free culture systems, such as a serum-free complete medium (VP-SFM), human platelet lysate and a synthetic trypsin (TrypLE™ Express); afterward, cell growth was assessed with the xCELLigence instrument. Animal-free products provided promising results, with performances similar or preferable to the common reagents; therefore their use could be encouraged for both ethical and technical advantages.

Autophagy processes are essential biological mechanisms control human cell survival and induction of cell death. Many researches indicate that cancer cells growth is affected by inhibition or induction of autophagy processes and laryngeal cancer "the most common type of head and neck cancer" is one of various types of tumors which effected by autophagy process. Numerous articles studied impact of adding autophagy inhibiter to cancer treatment protocol, and the current work study the in-vitro anticancer effect of doxorubicin alone and in combination with an autophagy inhibitor agent hydroxychloroquine against HEp-2 (laryngeal cancer) cell line. The present study suggested valuable effect of doxorubicin anticancer activity against HEp-2 cell line when used after hydroxychloroquine pretreatment, which may play promising role in treatment of laryngeal cancer. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors. IMPORTANCE Infection with the respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. Most RSV studies have involved either of two historic RSV/A strains infecting one of two cell lines, HEp-2 or A549 cells. However, RSV contains ample variation within two evolving subgroups (A and B), and HEp-2 and A549 cell lines are genetically distinct. Here, we measured viral action and host response in both HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporary strains. We discovered a subgroupdependent difference in viral gene expression and found A549 cells were more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings revealed important differences between RSV subgroups and two widely used cell lines and provided baseline data for experiments with model systems better representative of natural RSV infection [ABSTRACT FROM AUTHOR]

Aim. O6-methylguanine-DNA methyltransferase (MGMT) is an inducible repair enzyme that removes alkyl residues from DNA, thereby reducing the effectiveness of alkylating chemotherapy. Therefore, the MGMT inhibitors are used in medical practice. However, the most common of them, O6-benzylguanine and its analogs, have proven to be toxic to hematopoietic cells. That is why the search for new alternative inhibitors is essential. Methods. New nonnucleoside potential MGMT inhibitors were developed using semi-flexible docking. From an initial pool of 98 inhibitors, the most cytotoxic ones were screened out using the MTT-test and the clonogenic assay. Subsequently, a selected number of the inhibitors were tested alone and in combination with alkylating agent N-methyl-N′-nitro-Nnitrosoguanidine (MNNG) for investigation of the efficacy. This phase included clonogenic assay, Western blot analysis, and studies on their effects on autophagy and cell mortality in cancer cells in vitro using Monodansylcadaverine dying for autophagosomes and dying by Live-Dead Imaging Cell Kit respectively. In the final stage of the research, the efficacy of the combination therapy was evaluated using an in vivo ICR mice model. The tumor growth dynamics and changes in MGMT and other proteins level were investigated via Western blot. Results. Based on the data obtained, a number of new MGMT inhibitors exhibited low cytotoxicity and high efficacy at a concentration of 10 μM in vitro compared to O6-benzylguanine. Western blot analysis indicated that the inhibitors 41, 41B, 72, and 89 reduced MGMT level in the HEp-2 laryngeal carcinoma cells and T98G glioma cells compared to the controls. The combined treatment with the inhibitors and MNNG induced a high level of autophagy in the T98G cell line (glioma cells with highMGMT expression). However, a low level of autophagy was observed under the same conditions in U251MG cells (glioma cells lacking MGMT expression). The inhibitors did not increase the level of dead cells in either glioma cell line. Nevertheless, the combined treatment resulted in a high death rate exclusively in T98G cells. In vivo, the combined therapy with inhibitors 41, 41B, and 89 led to a significant reduction in tumor growth or remission, with inhibitor 89 more frequently achieving complete remission. Western blot analysis of treated tumors revealed decreased MGMT levels and increased cleaved caspase 3, suggesting apoptosis induction. Conclusions. The new low molecular weight non-nucleoside MGMT inhibitors effectively reduce MGMT protein levels and enhance the cytotoxic effects of MNNG across different cancer cell lines. In T98G cells, the combination treatment increases autophagy and makes cells more sensitive to MNNG. The in vivo study confirms the therapeutic potential of these inhibitors, with significant tumor reduction or remission observed. Inhibitor 41B demonstrated the greatest overall efficacy, making it a promising candidate for further development in the combined alkylating chemotherapy. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis (C. trachomatis) es una bacteria Gram negativa inmovil, se caracteriza por ser un microorganismo intracelular obligado y por poseer un ciclo reproductivo en el que puede distinguirse una forma infecciosa extracelular metabólicamente inerte (cuerpo elemental - EB’s), y una forma no infecciosa intracelular y activa (cuerpo reticulado - RB’s). C trachomatis se caracteriza por causar infección en humanos, está relacionada con enfermedades de transmisión sexual e infecciones oculares, por lo que puede conllevar a secuelas de interés si no se da un tratamiento oportuno. El objetivo de este estudio fue optimizar el modelo de infección de C. trachomatis en células HEp-2 con cuerpos elementales (EB’s) de C. trachomatis serovar L2. Inicialmente, se establecieron las condiciones para el crecimiento adecuado de las células HEp-2 en tiempo y con una confluencia del 90%, para continuar con la optimización de un protocolo de infección. La infección fue confirmada a partir de la coloración con Giemsa permitiendo evaluar características morfológicas tanto de las células HEp-2 sin infectar e infectadas y así mismo de los cuerpos elementales de C. trachomatis. Finalmente, se corroboró la infección con la técnica de Inmunofluorescencia directa que detecta la proteína de membrana MOMP de C. trachomatis. Tras los ensayos realizados se evidenció la presencia de cuerpos elementales próximos y dentro del citoplasma celular, así como células vacuoladas y daño celular causado por la infección.

Abstract Background Yes-associated protein (YAP) plays a crucial role in tumour development and it is the main effector of the Hippo signalling pathway. However, the mechanism underlying YAP downregulation in laryngeal cancer is still unclear. In our previous study, we found that YAP, compared with adjacent tissues, was expressed higher in laryngeal cancer and was also closely associated with histological differentiation, TNM stage and poor prognosis. Methods In this study, we attempted to determine whether silenced YAP could downregulate human laryngeal carcinoma Hep-2 cells progression. YAP was downregulated in Hep-2 cells by shRNA, and the malignant ability of Hep-2 was assessed in vitro and in vivo. Results In vitro, CCK-8, colony formation and wound healing assays showed that downregulation of YAP significantly reduced the rates of proliferation, migration, and invasion in Hep-2 cells. Downregulation of YAP distinctly induced G2/M cycle arrest and increased the rate of apoptosis. Accordingly, western blot assay suggested that the expression of DKK1, vimentin and β-catenin was significantly decreased after YAP downregulated treatment, thereby indicating that YAP mediated the EMT programme and the Wnt/β-catenin signalling pathway in carcinoma of the larynx. Furthermore, silencing of YAP suppressed Hep-2 cell tumourigenesis and metastasis in vivo. Conclusion In summary, our findings demonstrated the proliferation of YAP downregulation and the invasion of Hep-2 cells via downregulating the Wnt/β-catenin pathway in vitro and in vivo, suggesting that YAP may provide a potential therapeutic strategy for the treatment of laryngeal cancer.

The cytotoxic effect of catechol was examined in two human cancer cell lines, Epidermoid larynx carcinoma (Hep2), Cerebral glioblastoma multiforme (AMGM-5) and Murine mammary adenocarcinomacell (AMN3) treated with half concentrations of catechol (1000, 500, 250, 125, 62.5 and 32.25 µM) for 72 hr. The get hold of results showed catechol have a toxic effect of the cell viability of three types of cell lines after 72h of exposure, the toxicity was dependent on catechol concentrations and/or autoxidation for quinines formation, there were a marked decreased of cell viability in a dose dependent manner in all cell line types. Inhibition concentration of catechol for 50% of cell viability (IC50) were calculated, they were at 581.5µM, 478 µM and 833 µM of HEP-2, AMGM-5 and AMN3 cells, respectively. In addition the combination affects of three cell lines treated with catechol (previously treated at three concentrations close to IC50 were125, 250 and 500 µM for cell lines were incubated for 24 hr.) were assay with 100ul superoxide Dismutase (SOD), 500 ul peroxidase (POD) and theirs combination (100ul SOD and 500 ul POD) and 125 or 250 µg/mL of catechin against the toxicity of catechol in cell lines to estimated the reduction in quinine formation in these combinations, most inhibition rate of quinine formation display at 100ul SOD alone and combination with 500 ul POD in three cell lines in comparison to other treatments dependant to quinione formation in cells treated with catechol only. Higher percentage in inhibition rate of quinine formation were record in combination treatment of SOD & POD with three concentrations of catechol in AMGM5 cells (54.2%, 59.2% and 65%), followed Hep-2 (44.2%, 42.6% and 52.7%) and AMN3 in 38.7%, 46.6% and 50.7%, respectively, furthermore the less efficient protect obtained in treatment all cell lines with two concentrations of catechins. [ABSTRACT FROM AUTHOR]

The search for anti-nucleus antibodies (ANA) represents a fundamental step in the diagnosis of autoimmune diseases. The test considered the gold standard for ANA research is indirect immunofluorescence (IIF). The best substrate for ANA detection is provided by Human Epithelial type 2 (HEp-2) cells. The first phase of HEp-2 type image analysis involves the classification of fluorescence intensity in the positive/negative classes. However, the analysis of IIF images is difficult to perform and particularly dependent on the experience of the immunologist. For this reason, the interest of the scientific community in finding relevant technological solutions to the problem has been high. Deep learning, and in particular the Convolutional Neural Networks (CNNs), have demonstrated their effectiveness in the classification of biomedical images. In this work the efficacy of the CNN fine-tuning method applied to the problem of classification of fluorescence intensity in HEp-2 images was investigated. For this purpose, four of the best known pre-trained networks were analyzed (AlexNet, SqueezeNet, ResNet18, GoogLeNet). The classifying power of CNN was investigated with different training modalities; three levels of freezing weights and scratch. Performance analysis was conducted, in terms of area under the ROC (Receiver Operating Characteristic) curve (AUC) and accuracy, using a public database. The best result achieved an AUC equal to 98.6% and an accuracy of 93.9%, demonstrating an excellent ability to discriminate between the positive/negative fluorescence classes. For an effective performance comparison, the fine-tuning mode was compared to those in which CNNs are used as feature extractors, and the best configuration found was compared with other state-of-the-art works. [ABSTRACT FROM AUTHOR]

Cryopreservation was first studied in early twentieth century, and the cryopreservation of cell and tissue samples has become an inseparable process in biology research labs, helping scientists to store living materials and to accumulate specimens. Recently, a new and simplified cryopreservation product, BioFlash Drive™ SP developed by a US firm Fibulas, came to the attention of many researchers. It integrates the sample container with control-released cryoprotectant, and the container itself can achieve slow-freezing in deep freezers. This means no reagents mixing or extra steps for slow-freezing are needed. The design of this product aims to simplify the current cell-freezing procedures and reduce the lengthy and error prone processes. In this research, we compared the post-thaw cell viability using two of the most widely used protocols, with the one with BioFlash Drive™ SP (Fibulas). Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™). Results show there is not statistically significant difference in cell viability between the conventional protocols and the new protocols. However, this new protocol is less manual and less time consuming. With this new method, it might be possible for researchers to archive research progress more often, because saving cell can be an easier but still reliable experience. • The present research compares a novel cell cryopreservation kit and conventional cell freezing methods. • The results show that there is no significant difference among these methods. • The performance of the kit under non-usual extreme conditions is also studied. [ABSTRACT FROM AUTHOR]

Purpose: Development of pharmaceutical dosage forms of natural products has gained great interest recently. Propolis is a natural product with various active compounds and multiple pharmacological activities. Its resinous nature and low bioavailability were obstacles in the optimum use of this magnificent natural product. Aim: This study evaluates the effect of using liposomes as a drug delivery system on the enhancement of the cytotoxic effect of propolis on squamous cell carcinoma cell lines (Hep-2) of head and neck. Methods: An optimized liposomal formulation of propolis was prepared using the conventional thin film hydration method 1, 2. The prepared (Hep-2) cell line was treated with different concentrations of propolis and optimized propolis liposomes for 24 h. The effect of both propolis and propolis liposomes on cell line was investigated using MTT assay, cytological examination, and nuclear morphometric analysis. The effect of the drugs on the cell apoptosis was evaluated using Annexin V. Results: The findings revealed that both propolis and propolis liposomes have a cytotoxic effect on Hep-2 cell line through induction of apoptosis. The effect was dose dependent. However, a statistically significant enhancement in propolis-mediated apoptosis on Hep-2 cells was elucidated due to encapsulation within the prepared liposomes. Conclusion: Liposome is a powerful tool for enhancing the cytotoxicity of propolis against Hep-2 cell line.

Previous studies have provided evidence suggesting a role for apoptosis in the control of Herpes Simplex Virus 1 (HSV-1) latency. HSV-1 induces and then later blocks apoptosis in infected cells. The immediate early viral gene α0, which synthesizes the ICP0 protein, is necessary and sufficient for HSV-1-induced apoptosis in human epithelial (HEp-2) cells. While previous research showed that ICP0 protein synthesis is not necessary for HSV-1-induced apoptosis in infected HEp-2 cells, circumstantial evidence suggested that it might be needed in infected African green monkey kidney (Vero) cells. In this study, we determined the specific aspects of α0 needed to trigger apoptosis in these two cell types. HEp-2 cells transfected with α0 expressing plasmids that generated either full-length, truncated, or no detectable (multiple stop codons) ICP0 protein died through apoptosis. This indicates that ICP0 protein is not necessary for α0-induced apoptosis and that α0 mRNA alone has apoptotic induction properties in HEp-2 cells. We next investigated the primary structure of α0’s mRNA to better define its proapoptotic ability. Since α0 is one of the few HSV-1 genes that are spliced, we transfected cells with a plasmid expressing ICP0 from cDNA copy, pcDNAICP0. The cells transfected with pcDNAICP0 underwent apoptosis at a level equivalent to those transfected with the genomic copy of α0, which indicates that neither splicing events nor introns are required for the apoptotic function of α0 in HEp-2 cells. Next, we studied the ability of α0 to cause apoptosis in Vero cells. Since HSV-1-induced apoptosis in Vero cells requires protein synthesis early in infection, proteins synthesized with immediate early kinetics may facilitate apoptosis. Vero cells were transfected with plasmids producing either full-length ICP0 or ICP0 truncated at codon 212. Full-length ICP0, but not truncated ICP0, induced apoptosis in Vero cells. Together, these results suggest that α0 gene expression triggers apoptosis, but ICP0 protein is needed to facilitate apoptosis in Vero cells. In addition, ICP0’s facilitation activity may lie in its carboxyl-terminated domain. Thus, our results demonstrate that α0’s mRNA and protein possess proapoptotic properties. The requirement for ICP0 protein during HSV-dependent apoptosis appears to be cell type specific.

Chlamydia trachomatis (C. Trachomatis) is a Gram negative unmoving bacterium, characterized by being an obligate intracellular microorganism and having a reproductive cycle in which a metabolically inactive extracellular infectious form (elementary body - EB's) can be distinguished from an intracellular active and non-infectious form (reticulated body - RB's). C trachomatis is characterized by causing infection in humans, is related to sexually transmitted diseases and eye infections, so it can lead to sequelae of interest if timely treatment is not given. The objective of this study was to optimize the infection model of C. trachomatis in HEp-2 cells with elementary bodies (EB's) of C. trachomatis serovar L2. Initially, the conditions for the adequate growth of HEp-2 cells were established in time and with a confluence of 90%, to continue with the optimization of an infection protocol. The infection was confirmed from the staining with Giemsa allowing to evaluate morphological characteristics of both uninfected and infected HEp-2 cells and also of the elementary bodies of C. trachomatis. Finally, the infection was corroborated with the direct immunofluorescence technique, that detects the C. trachomatis MOMP membrane protein. After the tests were performed, the presence of elementary bodies nearby and within the cellular cytoplasm was evidenced, as well as vacuolated cells and cellular damage caused by the infection. [ABSTRACT FROM AUTHOR]

Antinuclear antibody (ANA) testing is best performed using the indirect immunofluorescence (IIF) method with human epithelial type-2 (HEp-2) cells as the substrate. IIF is a subjective procedure in which HEp-2 patterns are analyzed manually from the microscope. Therefore, ANA test results greatly rely on the experience and expertise of pathologists. Hence, complete automation of the ANA test is required to avoid incorrect diagnoses. This paper represents an algorithm for the complex HEp-2 cell classification problem. The proposed algorithm used a small hybrid feature set that characterizes the texture and morphology of the HEp-2 cells along with artificial neural network (ANN). The hybrid features were extracted by breaking up the image into eight binary images. The proposed hybrid descriptors were more efficient than the popular co-occurrence matrix descriptor and local binary pattern descriptors for texture analysis. The proposed algorithm was evaluated on the ICPR 2016 IIF HEp-2 cell image dataset. The results indicated that the hybrid descriptor with an ANN approach achieved improved performance, with "96.8%" mean class accuracy. [ABSTRACT FROM AUTHOR]

• First comprehensive work on in vitro antiviral assay using HHV-3 from Chickenpox and Herpes Zoster patients. • In vitro cytotoxicity assay of Aqueous, Ethanolic & aqueous ethanolic leaf extracts of chosen plant leaves in HEp-2 cells. • First of its kind in reporting the in vitro antiviral activity in the leaves of chosen plants in comparison with Acyclovir against the HHV-3. • First of its kind in in silico analysis of the phytochemicals from Punica granatum L. against the HHV-3 protease through discovery studio. • Interpretation of ADMETSAR properties of the screened active phytochemicals. Human Herpes Virus-3 (Varicella Zoster Virus) causes Chickenpox in childhood and reactivates after decades of being latent to cause Herpes Zoster in adults. The aim of this study was to evaluate the in vitro antiviral potency on the leaves of Punica granatum L., Momordica charantia L., Andrographis paniculata Nees. and Melia azedarach L., against the Human Herpes Virus-3 isolated from Chickenpox and Zoster in comparison with acyclovir. Aqueous, ethanolic and aqueous ethanolic extracts were prepared from the chosen plant leaves by lyophilization process and subjected to in vitro cytotoxicity assay in HEp-2 cells followed by the in vitro antiviral evaluation against the clinical isolates of HHV-3 using post incubation assay. The structure of leaf chemicals were retrieved from protein data bank and in silico drug analysis was carried out through discovery studio targeting the protease of HHV-3.The drug likeliness and the ADMETSAR properties of the screened active phytochemicals were calculated. Aqueous extract from the leaves of Punica granatum L., exhibited potential antiviral activity against the HHV-3. The in silico docking results found that the phytochemicals of Punica granatum L., interacted on the active site of the HHV-3 protease. Aqueous extract from the leaves of Punica granatum L. was superior in exhibiting its antiviral efficacy to HHV-3 whose in vitro activity was comparable with acyclovir. As the leaf phytochemicals interacted with the HHV-3 protease, the antiviral activity of the Punica granatum L., leaves may interfere with the capsid assembly of the HHV-3. [ABSTRACT FROM AUTHOR]

Previous studies have provided evidence suggesting a role for apoptosis in the control of Herpes Simplex Virus 1 (HSV-1) latency. HSV-1 induces and then later blocks apoptosis in infected cells. The immediate early viral gene α0, which synthesizes the ICP0 protein, is necessary and sufficient for HSV-1-induced apoptosis in human epithelial (HEp-2) cells. While previous research showed that ICP0 protein synthesis is not necessary for HSV-1-induced apoptosis in infected HEp-2 cells, circumstantial evidence suggested that it might be needed in infected African green monkey kidney (Vero) cells. In this study, we determined the specific aspects of α0 needed to trigger apoptosis in these two cell types. HEp-2 cells transfected with α0 expressing plasmids that generated either full-length, truncated, or no detectable (multiple stop codons) ICP0 protein died through apoptosis. This indicates that ICP0 protein is not necessary for α0-induced apoptosis and that α0 mRNA alone has apoptotic induction properties in HEp-2 cells. We next investigated the primary structure of α0's mRNA to better define its proapoptotic ability. Since α0 is one of the few HSV-1 genes that are spliced, we transfected cells with a plasmid expressing ICP0 from cDNA copy, pcDNAICP0. The cells transfected with pcDNAICP0 underwent apoptosis at a level equivalent to those transfected with the genomic copy of α0, which indicates that neither splicing events nor introns are required for the apoptotic function of α0 in HEp-2 cells. Next, we studied the ability of α0 to cause apoptosis in Vero cells. Since HSV-1-induced apoptosis in Vero cells requires protein synthesis early in infection, proteins synthesized with immediate early kinetics may facilitate apoptosis. Vero cells were transfected with plasmids producing either full-length ICP0 or ICP0 truncated at codon 212. Full-length ICP0, but not truncated ICP0, induced apoptosis in Vero cells. Together, these results suggest that α0 gene expression triggers apoptosis, but ICP0 protein is needed to facilitate apoptosis in Vero cells. In addition, ICP0's facilitation activity may lie in its carboxyl-terminated domain. Thus, our results demonstrate that α0's mRNA and protein possess proapoptotic properties. The requirement for ICP0 protein during HSV-dependent apoptosis appears to be cell type specific. [ABSTRACT FROM AUTHOR]

Graphical abstract Four mixed-ligand Cu(II) complexes with 1,10-phen/2,2′-bipy and tetrazole derivatives are reported together with a comparative study of their properties and cytotoxic activity (on Hep-2 and MCF-7 cancer cell lines). Highlights • Mixed-ligand Cu(II) complexes with diimine and tetrazole derivatives were synthesized. • Crystal structures were determined using single-crystal X-ray diffraction analysis. • Fluorescent microscopy was employed to examine the cytotoxic and apoptotic effects. • The complexes showed cytotoxic activity in vitro against MCF-7 and Hep-2 cell lines. Abstract The [Cu 2 (2,2′-bipy) 2 (L1) 4 ] (1), [Cu 2 (1,10-phen) 2 (L1) 4 ] (2), [Cu(2,2′-bipy)(L2) 2 ] n (3) and [Cu 2 (1,10-phen) 2 (L2) 4 ] (4) complexes, where HL1 – 5-phenyltetrazole, and HL2 – 1 H -tetrazole, have been synthesized. All complexes have been characterized by elemental analysis, IR, EPR spectroscopy and X-ray diffraction. The complexes possess distorted tetragonal-pyramidal coordination geometry. Compounds 1 , 2 , 4 show μ-5-phenyl-tetrazole/tetrazole bridged dinuclear structures, while compound 3 reveals polymeric structure. The effect of compounds on viability of the MCF-7 and Hep-2 cell lines was investigated in vitro. The study showed that tetrazole ligands HL1 and HL2 are non-toxic at tested concentrations (1–50 μM), while 1,10-phen and 2,2′-bipy posses cytotoxicity. All of the complexes exhibit significant dose-dependent cytotoxic effect and have the potential to act as efficient cytotoxic drugs. The complexes [Cu(1,10-phen)Cl 2 ] (5) and [Cu(2,2′-bipy)Cl 2 ] (6) have also been obtained to establish the influence of insertion of tetrazole ligands in compounds on their cytotoxic properties. [ABSTRACT FROM AUTHOR]

The search for anti-nucleus antibodies (ANA) represents a fundamental step in the diagnosis of autoimmune diseases. The test considered the gold standard for ANA research is indirect immunofluorescence (IIF). The best substrate for ANA detection is provided by Human Epithelial type 2 (HEp-2) cells. The first phase of HEp-2 type image analysis involves the classification of fluorescence intensity in the positive/negative classes. However, the analysis of IIF images is difficult to perform and particularly dependent on the experience of the immunologist. For this reason, the interest of the scientific community in finding relevant technological solutions to the problem has been high. Deep learning, and in particular the Convolutional Neural Networks (CNNs), have demonstrated their effectiveness in the classification of biomedical images. In this work the efficacy of the CNN fine-tuning method applied to the problem of classification of fluorescence intensity in HEp-2 images was investigated. For this purpose, four of the best known pre-trained networks were analyzed (AlexNet, SqueezeNet, ResNet18, GoogLeNet). The classifying power of CNN was investigated with different training modalities; three levels of freezing weights and scratch. Performance analysis was conducted, in terms of area under the ROC (Receiver Operating Characteristic) curve (AUC) and accuracy, using a public database. The best result achieved an AUC equal to 98.6% and an accuracy of 93.9%, demonstrating an excellent ability to discriminate between the positive/negative fluorescence classes. For an effective performance comparison, the fine-tuning mode was compared to those in which CNNs are used as feature extractors, and the best configuration found was compared with other state-of-the-art works.

The aim of the present study was to determine whether acetazolamide (Ace) treatment enhances the chemosensitivity of Hep‑2 laryngeal cells to cisplatin (Cis). At the logarithmic growth phase, Hep‑2 cells were treated with Ace, Cis or both, and cell viability was detected using an MTT assay. The degree of apoptosis was detected using flow cytometry. Expression levels of apoptosis‑related proteins, including BCL2 apoptosis regulator (bcl‑2), BCL2 associated X (bax) and caspase‑3, and of proliferation‑related proteins, including proliferating cell nuclear antigen (PCNA) and tumor protein p53 (P53), were detected using western blotting. mRNA expression levels of aquaporin‑1 (AQP1) in each group were detected using reverse transcription‑polymerase chain reaction. Compared with the drugs used alone, treatment with both Ace and Cis displayed synergistic effects on the growth inhibition and apoptosis induction in Hep‑2 cells. The Ace/Cis combination decreased the expression of PCNA but increased the expression of p53. In addition, the combination treatment decreased the ratio of bcl‑2/bax and increased the expression of caspase‑3, as well as decreased the expression of AQP1. These results demonstrated that the combined use of Ace and Cis enhanced the chemosensitivity of laryngeal carcinoma cells. [ABSTRACT FROM AUTHOR]

Aspirin may exhibit antitumor activities, as it is able to inhibit cell proliferation. However, the ability of aspirin to inhibit cellular proliferation in Hep‑2 cells and its underlying molecular mechanisms have been poorly determined. The aim of the present study was to investigate whether aspirin may induce cell apoptosis in the neoplastic cell line Hep‑2. The effects of aspirin on the migratory and invasive abilities of Hep‑2 cells were also investigated using Transwell assays. In the present study, it was demonstrated that aspirin induced apoptosis and inhibited proliferation, migration and invasion in Hep‑2 cells. Aspirin also significantly decreased the expression of B‑cell lymphoma 2 (Bcl‑2) and caspase‑3, and increased the expression of Bcl‑2‑associated X protein, suggesting that aspirin induced apoptosis through the intrinsic apoptotic pathway. Hep‑2 cells treated with aspirin exhibited a significant upregulation of phosphatase and tensin homolog (PTEN) and decreased levels of phosphorylated protein kinase B (AKT). However, the total amount of AKT protein was not altered in response to aspirin treatment. Furthermore, the expression of nuclear factor (NF)‑κB and survivin, which are the downstream targets of the PTEN/AKT signaling pathway, was inhibited. These results indicated that the molecular mechanism underlying the antitumor effects of aspirin may be associated with the inhibition of tumor invasion and induction of apoptosis by regulating the activity of the PTEN/AKT/NF‑κB/survivin signaling pathway. [ABSTRACT FROM AUTHOR]

Aim: infection is an important global health problem in developing countries where hygiene is poor and hence shigellosis is a main cause of diarrhoea-associated mortality and morbidity, particularly in children under the age of five. The bacterial entry into colon and rectal epithelial cells has been named ‘bacterium-directed phagocytosis’. This term highlights that the bacteria actively stimulate their own uptake into non-professional phagocytes. The aim of this study was to demonstrate the invasion of HEp-2 cells by spp. isolated from acute pediatric diarrhea in Tehran, Iran.Methods: Three-hundred and ten non-duplicative diarrheal stool samples were collected from the children admitted to Children’s Medical Center in Tehran, Iran. Samples were cultured and suspected colonies were identified by routine microbiological and biochemical tests. The invasion of the two isolated spp. to HEp-2 cells was studied.Results: Of 310 stool samples, 16 (5.2%) spp. were isolated, including seven (43.7%) and nine (56.3%) . Four (44.4%) and seven (42.8%) showed invasive phenotype to HEp-2.Conclusion: and are reported as the most prevalent spp. in nature which infect humans. Invasion of various cell lines gives the chance of survival to spp. This ability causes more virulent infections in the host. Despite costly and time consuming cell culture techniques, the current method described in this paper is reliable for detecting invasive behavior of spp. Results have also shown that not all the spp. are able to invade intestinal epithelial cells.

Recently there has been an increase demand for Computer-Aided Diagnosis (CAD) tools to support clinicians in the field of Indirect ImmunoFluorescence (IIF), as the novel digital imaging reading approach can help to overcome the reader subjectivity. Nevertheless, a large multicenter evaluation of the inter-observer reading variability in this field is still missing. This work fills this gap as we evaluated 556 consecutive samples, for a total of 1679 images, collected in three laboratories with IIF expertise using HEp-2 cell substrate (MBL) at 1:80 screening dilution according to conventional procedures. In each laboratory, the images were blindly classified by two experts into three intensity classes: positive, negative, and weak positive. Positive and weak positive ANA-IIF results were categorized by the predominant fluorescence pattern among six main classes. Data were pairwise analyzed and the inter-observer reading variability was measured by Cohen's kappa test, revealing a pairwise agreement little further away than substantial both for fluorescence intensity and for staining pattern recognition (k = 0.602 and k = 0.627, respectively). We also noticed that the inter-observer reading variability decreases when it is measured with respect to a gold standard classification computed on the basis of labels assigned by the three laboratories. These data show that laboratory agreement improves using digital images and comparing each single human evaluation to potential reference data, suggesting that a solid gold standard is essential to properly make use of CAD systems in routine work lab. [ABSTRACT FROM AUTHOR]

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Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A–L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways. [ABSTRACT FROM AUTHOR]

The nanomaterial with the novel biologically active compounds has been actively investigated for application in cancer research. Substantial use of nanofibrous scaffold for cancer research with potentially bioactive compounds through electrospinning has not been fully explored. Here, we describe the series of fabrication of nanofibrous scaffold loaded with novel potential biologically active hydroxybenzo[ a ]phenazine pyrazol-5(4H)-one derivatives were designed, synthesized by a simple one-pot, two step four component condensation based on Michael type addition reaction of lawsone, benzene-1,2-diamine, aromatic aldehydes and 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one as the substrates. The heterogeneous solid state catalyst (Fe (III) Y-Zeolite) could effectively catalyze the reaction to obtain the product with high yield and short reaction time. The synthesized compounds (5a–5p) were analyzed by NMR, FTIR and HRMS analysis. Compound 5c was confirmed by single crystal XRD studies. All the compounds were biologically evaluated for their potential inhibitory effect on anticancer (MCF-7, Hep-2) and microbial (MRSA, MTCC 201 and FRCA) activities. Among the compounds 5i exhibited the highest levels of inhibitory activity against both MCF-7, Hep-2 cell lines. Furthermore, the compound 5i (BPP) was evaluated for DNA fragmentation, flow cytometry studies and cytotoxicity against MCF-7, Hep-2 and NIH 3T3 fibroblast cell lines. In addition, molecular docking (PDB ID: 1T46 ) studies were performed to predict the binding affinity of ligand with receptor. Moreover, the synthesized BPP compound was loaded in to the PHB-PCL nanofibrous scaffold to check the cytotoxicity against the MCF-7, Hep-2 and NIH 3T3 fibroblast cell lines. The in vitro apoptotic potential of the PHB-PCL-BPP nanofibrous scaffold was assessed against MCF-7, Hep-2 cancerous cells and fibroblast cells at 12, 24 and 48 h respectively. The nanofibrous scaffold with BPP can induce apoptosis and also suppress the proliferation of cancerous cells. We anticipate that our results can provide better potential research in nanomaterial based cancer research. [ABSTRACT FROM AUTHOR]

Simple chemical co-precipitation method has been employed to synthesize two dimensional copper (Cu) doped tungsten oxide (WO 3 ) nanoplates. A numbers of characterization techniques have been used to investigate their structural, optical and biocompatible anti cancer properties. The XRD results have confirmed the monoclinic crystal structure of WO 3 nanoplates, and also successful doping of Cu ions into the WO 3 crystal lattice. The presence of functional groups and chemical bonding have been verified through FTIR and Raman spectroscopy. The SEM images demonstrate that both undoped and Cu doped WO 3 samples have squares plate like morphology. The EDX spectra confirm the presence of Cu, W and O ions. Diffuse reflectance spectroscopy (DRS) analysis has revealed a substantial red-shift in the absorption edge and a decrease in the band gap energy of nanoplates with Cu doping. Photoluminescence spectroscopy has been used to study the presence of defects like oxygen vacancies. Furthermore, the differential cytotoxic properties of Cu doped WO 3 samples have been evaluated against human breast (MCF-7) and liver (Hep-2) cancer cells with ectocervical epithelial (HECE) healthy cells. The present findings confirm that the Cu doped WO 3 nanoplates can be used as an efficient biocompatible anti cancer agent. [ABSTRACT FROM AUTHOR]

Nanotechnology is an emerging field in science and technology, which can be applied to synthesize new materials at the nanoscale level. The present investigation aimed at comparing the synthesis, characterization and in vitro anticancer efficacy of synthesized silver and gold nanoparticles using leaves extract of Bauhinia tomentosa Linn. Silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) were synthesized using aqueous extract of leaves with solution of silver nitrate (AgNO, 1 mM) and chloroauric acid (HAuCl⋅3HO, 1 mM), respectively. The synthesized nanoparticles were characterized using UV-visible spectrophotometry, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, high-resolution transmission electron microscopy, energy-dispersive analysis of X-rays, X-ray diffraction, thermogravimetric analysis and cyclic voltammetry, which confirmed the reduction of Ag ions to Ag and Au ions to Au. The in vitro anticancer efficacy of AgNPs, AuNPs and aqueous extract of leaves confirmed by MTT assay exhibited IC concentrations of 28.125, 46.875 and 50 μg ml for lung A-549 cells, 103.125, 34.375 and 53.125 μg ml for HEp-2 cells and 62.5, 23.4 and 13.26 μg ml for MCF-7 cells, respectively. The concentrations indicate that both silver and gold nanoparticles as well as aqueous extract of leaves exhibited high anticancer efficacy. [ABSTRACT FROM AUTHOR]

Two bis(tetrahydroisoquinoline) derivatives of undecane were synthesized from brassylic acid and 3,4-dimethoxy-(or methylenedioxy-)phenylethylamine. It was shown that 4a and 4b were highly cytotoxic for cancer-cell cultures and less toxic to healthy cells and exhibited noticeable antimicrobial activity against Gram-positive and Gram-negative bacteria and fungal strain Candida albicans. The antifungal activity of 4a exceeded that of the reference drug nystatin. [ABSTRACT FROM AUTHOR]

Antinuclear antibodies (ANA) are important diagnostic markers in many autoimmune rheumatological diseases. The indirect immunofluorescence assay applied on human epithelial cells generates images that are used in the detection of ANA. The classification of these images for different ANA patterns requires human experts. It is time-consuming and subjective as different experts may label the same image differently. Therefore, there is an interest in machine learning-based automatic classification of ANA patterns. In our study, to build an application for the automatic classification of ANA patterns, we construct a dataset and learn a deep neural network with a transfer learning approach. We show that even in the existence of a limited number of labeled data, high accuracies can be achieved on the unseen test samples. Our study shows that deep learning-based software can be built for this task to save expert time., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Boral et al.)

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells.

In the present study, we investigated the mechanisms of chamaejasmine action on human HEp-2 larynx carcinoma cells, which possess constitutively active Akt. Results indicated that chamaejasmine showed more notable anticancer activity than apigenin against HEp-2, PC-3, NCI-H1975, HT-29 and SKOV-3. Moreover, chamaejasmine presented most significantly inhibition towards HEp-2, with IC50 values of 1.92 µM. Treatment of HEp-2 cells with chamaejasmine (1–4 μM) resulted in significant dose-dependent decrease in Akt phosphorylation at Serine473. Chamaejasmine-mediated dephosphorylation of Akt resulted in inhibition of its kinase activity, which was confirmed by reduced phosphorylation of proapoptotic proteins BAD and glycogen synthase kinase-3, essential downstream targets of Akt. Inactivation of Akt seems to be associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation upon chamaejasmine treatment. Exposure to chamaejasmine significantly induced caspase-9 and caspase-3 activity. In vivo, chamaejasmine intake through gavage resulted in inactivation of Akt and induction of apoptosis in HEp-2 tumors. These results suggest that Akt inactivation and dephosphorylation of BAD is a critical event, at least in part, in chamaejasmine-induced HEp-2 cells apoptosis.

According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight ( n = 84), Helios ( n = 85) and NOVA View ( n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A ( k = 0.82) and B ( k = 0.72), and almost perfect for C ( k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C ( k = 0.86; k = 0.82) and substantial agreement for A ( k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform. [ABSTRACT FROM AUTHOR]

Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing antiadherence modalities for preharvest control of O157 in cattle. [ABSTRACT FROM AUTHOR]

Background: Alkhumra hemorrhagic fever virus (AHFV) is a flavivirus that was discovered in 1995 in Saudi Arabia. Clinical manifestations of AHFV infection include hemorrhagic fever, hepatitis, and encephalitis with a reported mortality rate as high as 25%. There are no published data on the growth characteristics of AHFV in mammalian cell lines. The objective of this study was to examine the ability of AHFV to grow and propagate in four of the commonly used mammalian cell culture lines and to determine the virus growth curve characteristics in each. Materials and Methods: Human epidermoid carcinoma (HEp-2), LLC-MK2, Madin-Darby canine kidney (MDCK), and Vero cell lines were inoculated with AHFV. The virus production by each cell line was determined by growth curve studies. Mean titers were calculated and expressed as median tissue culture infective dose per mL (TCID50/mL). Results: AHFV grew and propagated to variable titers in the employed cell lines. The highest mean titers were observed in the LLC-MK2, followed by the MDCK, Vero, and HEP-2, in descending order. Conclusions: The growth curve studies showed that AHFV can propagate in the four types of cell lines to variable titers. LLC-MK2 cells are superior to MDCK, Vero, and HEP-2 for propagation of AHFV. [ABSTRACT FROM AUTHOR]

Enteroaggregative Escherichia coli (EAEC) bears remarkable capacity to adhere the host intestinal mucosal surface and results in acute or persistent childhood diarrhea worldwide. In this study, an attempt has been made to inhibit EAEC cell adherence in-vitro using synthetic peptides. E. coli isolates (n = 54) were isolated from the stool samples of clinically diagnosed pediatric diarrheal patients. 92.8% isolates showed different types of aggregative adherence patterns with HEp-2 cells. AAF-II (Aggregative Adherence Fimbriae-II) EAEC exhibited the maximum ability to form biofilm and intracellular survival. Peptides were designed against the high antigenic epitopic regions of AAF-II adhesin of EAEC O42 using prediction algorithms like BcePred and ProPred software to block the EAEC cell adhesion in-vitro . Peptides P2 (DITITPATNRDVNV) and P3 (MRIKAWGEANHGQL) demonstrated higher inhibition of EAEC cell adhesion than P1 (GMQGSITPAIPLRPG). Interestingly, increasing the pre-incubation time of the peptides with HEp-2 cells from 1 h to 2 h showed the maximum inhibition. The data suggested the potential role of P2 and P3 peptides in successfully blocking the binding of AAF-II EAEC with HEp-2 cell receptors. Hence, the peptides may be efficacious in designing new chemotherapeutic for the management of EAEC mediated diarrhea. [ABSTRACT FROM AUTHOR]

Genistein and trichostatin A (TSA) are two chemotherapeutic compounds with antitumor effects in different types of cancer cell. However, the effects of genistein and TSA on the HEp-2 laryngeal cancer cell line remain to be fully elucidated. In the present study, it was found that genistein and TSA inhibited cell growth and cell migration, and promoted apoptosis in the HEp-2 laryngeal cancer cell line. The HEp-2 cells were treated with genistein, TSA or the two compounds in combination. Cell proliferation and apoptosis were measured using an MTT assay, Annexin V/propidium iodide staining and a TUNEL assay. Cell invasion was determined using a Matrigel-based Transwell assay. Western blotting was used to examine the activation of the Akt pathway and the expression levels of pro-or anti-apoptotic proteins. Treatment with either genistein or TSA alone mildly inhibited cell viability, growth and invasion, and induced the apoptosis of the laryngeal cancer cells, whereas more marked effects were observed in the cells treated with the combination of the two compounds. In addition, genistein reversed endothelial growth factor-induced epithelial-mesenchymal transition (EMT) in the HEp-2 cells, the effect of which were was further increased by joint application with TSA. Treatment of the HEp-2 cells with genistein and TSA led to a significant reduction in the phosphorylation of Akt and activation of its downstream target, and resulted in peroxisome proliferator-activated receptor-y cleavage, increased expression of B cell lymphoma-2 (Bcl-2)-associated X protein and reduced the expression of Bcl-2. In conclusion, the present study demonstrated that, with the involvement of TSA, genistein exhibited substantial advantages in inhibiting laryngeal carcinoma cell growth, invasion and EMT, and induced apoptosis, compared with genistein treatment alone, which occurred through the regulation of Akt activation and the apoptotic pathway. [ABSTRACT FROM AUTHOR]

Purpose The purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. Material and methods PLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48 h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. Results The drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. Conclusion Curcumin nanoparticles could be more active in inducing apoptosis in short term assays (24 h) than long term assays (48 h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48 h) compared to curcumin group. [ABSTRACT FROM AUTHOR]

It has been previously reported that cisplatin is a well-known anticancer drug being used against a wide range of malignancies including head and neck, ovarian and non-small cell lung carcinoma, and demonstrated its anticancer activity by reacting with DNA or changing cell structure, immune response, reactive oxygen species level (ROS). In this research we proved that cisplatin induced cell injuries and heme oxygenase-1 (HO-1) expression in laryngeal squamous cell cancer Hep-2 cells through ROS generation. The induction of HO-1 clearly protected Hep-2 cells from cisplatin-induced cell death and ROS reaction, and the inhibitor of HO-1 enhanced the cell death and ROS generation induced by cisplatin. Furthermore, the HO-1 expression induced by cisplatin was strongly inhibited by the knockdown of nuclear factor-erythroid-2-related factor-2 (Nrf-2), and the oxidative damages induced by cisplatin were significantly enhanced. Therefore, it may be concluded that the inhibition of HO-1 or the knockdown of Nrf-2 significantly enhanced cisplatin's anticancer effects on Hep-2 cells. In clinic, with the overexpression of HO-1 in laryngeal squamous cancer tissues, the combination of cisplatin with the inhibitor of HO-1 or Nrf-2 siRNA may act as a new method to the treatment of laryngeal squamous cancer. [ABSTRACT FROM AUTHOR]