Cell Line T84 Research Articles

Preparation and evaluation of peptide-dendrimer-paclitaxel conjugates for treatment of heterogeneous stage 1 nonsmall cell lung cancer in 293T and L132 cell lines

Purpose: To develop peptide-dendrimer-paclitaxel conjugates for the treatment of heterogeneous stage 1 non small cell lung cancer (NSCLC) in 293T and L132cell line. Method: Dendrimer-paclitaxel conjugates (PAMAM-PTX) were prepared by NHS method and the conjugates were used for the synthesis of peptide-dendrimer-paclitaxel conjugates (GE-PAMAM-PTX). The particle sizes of PAMAM-PTX and GE-PAMAM-PTX were measured. Entrapment efficiency of PTX in PAMAM-PTX was measured while GE-PAMAM-PTX. PTX release from PAMAM-PTX and GEPAMAM- PTX was determined using a dialysis bag in pH 7.4 phosphate buffer. The cytotoxicity of PAMAM-PTX, GE-PAMAM-PTX, PAMAM and PTX was evaluated by 3-(4,5-dimethylthiazol-2-Yl)-2,5- diphenyltetrazolium bromide (MTT) assay using 293T cell lines. In vitro cellular uptake assay of PAMAM-PTX and GE-PAMAM-PTX and PTX at concentrations ranging from 0.01 to 0.5μM for 8 h was carried out in NSCLC cell lines 293T and L132. Results: More than 95 % entrapment efficiency of GE-PAMAM-PTX was observed with loading efficiency of 25 %. GE-PAMAM-PTX conjugates showed sustained release of PTX (~85 %) towards the end of 50 h. GE-PAMAM-PTX conjugates were more cytotoxic than pure PTX and PAMAM-PTX conjugates. The remarkable uptake of GE-PAMAM-PTX appear to be due to receptor-mediated endocytosis in the cell lines. The presence of ligand (GE) on PAMAM-PTX surface enabled the complex to bind to the over-expressed receptors on the cell lines. Conclusion: GE-PAMAM-PTX can facilitate targeting of paclitaxel to lung cancer cell lines and tumors and facilitate release of the drugs in a sustained manner to improve the therapeutic efficacy of PTX. Keywords: Paclitaxel, Lung cancer, Non-small cell lung cancer, Dendrimer, Peptide, PAMAM

Construction and identification of lentiviral vector mediated GATA-3 gene RNA interference in mouse

Objective:The aim of this study was to construct and identify a lentiviral vector for RNA interference of mouse GATA-3.Method:The coding region of the GATA-3 gene sequences were analyzed for a single-stranded oligo design and synthesis.PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the lentiviral vector and transformed into the competent cell DH5α.An expression vector shRNA-GATA-3 was constructed after sequencing and identifying the gene sequence of the recombinant clones.Accoding to the target gene sequence ,four pairs are as following: siRNA-685 group(LV3-GATA-3-Mus-685),siRNA-1152 group (LV3-GATA-3-Mus-1152),siRNA-1615 group(LV3-GATA-3-Mus-1615) and control group ,which were designed and syntheized and builded into the lentiviral carrier.The shRNA vectors were cotransfected with expression vectors into the 293T cell line,and the optimal interference sequence was screened by QPCR. The 293T cells were cotranfected with the constructed lentiviral interference vectors and packing plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. Result:Gene sequence proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The relative expression of GATA-3 mRNA in siRNA-1615 group(0.004) was significantly reduced after RNA interference comparing with that in the control group(0.022),siRNA-1152 group(0.009) and siRNA-685 group(0.009). The screened LV3-GATA-3-Mus-1615 ,as the most effective interference sequence,was constructed into the lentiviral vector.The titer of the concentrated virus of LV3-GATA-3-Mus-1615 was 5×108 TU/ml in the viral supernate of 293T cells collected at 48 hours after co-transfection. Conclusion:Alentiviral RNAi expression vector targeting the GATA-3 gene (LV3-GATA-3-Mus-1615) was successfully constrcted and identified.

Upregulation of MiR-369-3p suppresses cell migration and proliferation by targeting SOX4 in Hirschsprung's disease

BackgroundHirschsprung disease (HSCR) is a congenital digestive disease in the new born. miR-369-3p has been reported to be involved in many human diseases. However, the relationship between miR-369-3p and HSCR remains largely unknown. MethodsIn this study, qRT-PCR was used to detect the relative expression of miR-369-3p in 60 HSCR bowel tissue samples and 47 matched controls. Bioinformatic analysis and dual-luciferase reporter assay were performed to evaluate the target for miR-369-3p. Cell Counting Kit-8 (CCK-8) assay, Transwell assay, wound healing assay and flow cytometry were employed to investigate the biological function of miR-369-3p in human SH-SY5Y and 293T cell lines. ResultsWe found that ganglion cell numbers were remarkably reduced while miR-369-3p was significantly upregulated in HSCR tissues compared to that in adjacent normal tissues (P<0.01). Dual-luciferase reporter assay showed that the 3′-UTR of SOX4 was a direct target to miR-369-3p. Moreover, an increased level of miR-369-3p was inversely correlated with decreased levels of SOX4 mRNA and protein (P<0.05, respectively). Dysregulation of miR-369-3p and SOX4 significantly suppressed cell proliferation and migration in SH-SY5Y and 293T cell lines in vitro (P<0.05, respectively). ConclusionOur study demonstrates that aberrant expression of miR-369-3p might play a crucial role in the development HSCR by regulating SOX4 expression, which may infer that it is an effective diagnostic target in the pathogenesis of HSCR, but investigation is still needed to explore the underlying mechanism.

Induction of Osteopontin by Dengue Virus-3 Infection in THP-1 Cells: Inhibition of the Synthesis by Brefelamide and Its Derivative.

Osteopontin (OPN) is a multifunctional matricellular protein produced by a broad range of cells including osteoclasts, macrophages, T cells, endothelial cells, and vascular smooth muscle cells. OPN modulates various physiological and pathological events such as inflammation, wound healing, and bone formation and remodeling. Dengue virus (DENV) infection causes an increase in plasma OPN levels, which is correlated with the severity of symptoms and coagulation abnormalities. DENV infection also induces OPN gene expression in human macrophages. This study investigated the inhibitory effects of brefelamide and its methyl ether derivative on DENV-3 by measuring changes in OPN levels in human THP-1 and 293T cell lines infected at different multiplicities of infection and post-infection time points. OPN mRNA expression and viral RNA were detected by reverse transcriptase quantitative real-time PCR, whereas protein level was determined by enzyme-linked immunosorbent assay. We found that viral copy number was higher in 293T than in THP-1 cells. However, THP-1 constitutively expressed higher levels of OPN mRNA and protein, which were enhanced by DENV-3 infection. Brefelamide and its derivative suppressed OPN production in DENV-3 infected THP-1 cells; the effective doses of these compounds had no effect on uninfected cells, indicating low cytotoxicity. These results suggest that brefelamide and its methyl ether derivative have therapeutic effects in preventing inflammation, coagulopathy, and fibrinolysis caused by OPN upregulation induced by DENV-3 infection.

Role of DNA methylation in expression control of the IKZF3-GSDMA region in human epithelial cells.

Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site’s methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8–13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation.

Facile and efficient access to Androsten-17-(1′,3′,4′)-pyrazoles and Androst-17β-(1′,3′,4′)-pyrazoles via Vilsmeier reagents, and their antiproliferative activity evaluation in vitro

In this work, twenty-seven novel steroidal pyrazole derivatives were designed and effectively synthesized with two different commercially available staring material, Isopregnanolone 1 and 5,16-Pregnadienolone 7, via the key intermediates, 17β-(4′-formyl)pyrazolylandrost-3β-yl formate and 17-(4′-formyl)pyrazolylandrost- 5,16-dienes-3β-yl formate, which were obtained from the cyclization of steroidal phenylhydrazone with Vilsmeier reagent catalyzed by phosphorous oxychloride followed by hydrolysis, then Borch reduction to afford the target derivatives under mild conditions. Structures of these compounds were identified by 1H NMR, 13C NMR and high resolution mass spectrometry. Based on our previous work, the cytotoxicity of these derivatives were evaluated by the SRB method against 293T cell lines and three cancer cell lines: A549, Hela and MCF-7. The results indicated that compounds 5b-d, and 11a-e exhibited moderate to high cytotoxic activities with IC50 values ranging from 0.62 to 7.51 μM. Among the eight hybrids, compound 11b, with an ethyl amino and a dien-pregn moieties showed the highest potency, with an IC50 values of 0.87 μM and 0.53 μM for 293T cell lines and Hela cell lines, respectively. Some structure-activity relationships among the groups of the twenty-seven derivatives are discussed and identify several determinants important for the activity of these compounds. What's more, further molecular mechanism studies suggested that 11b one of the most potent derivatives caused Hela cell lines apoptosis and arrested the cell cycle at S phase in a concentration dependent manner.

Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis

A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

Overexpression of KAI1/CD82 suppresses invitro cell growth, migration, invasion and xenograft growth in oral cancer.

KAI1/CD82 is a metastatic suppressor gene in human prostate cancer and several other types of cancer in humans. The present study aimed to examine the role of the overexpression of KAI1 in the progression of oral cancer. Human KAI1/CD82 cDNA was transfected into OSCC-15 and 293T cell lines, and its effects on OSCC-15 cell proliferation, invasion and apoptosis were assessed by performing a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, Matrigel invasion and Annexin V-FITC staining, respectively. In addition, a xenograft model was used to assess the effect of KAI1/CD82 on the in vivo growth of tumors. The overexpression of KAI1/CD82 inhibited the proliferation and invasion of OSCC-15 cells. It also enhanced the apoptotic rate of the OSCC-15 cells. Furthermore, the overexpression of KAI1/CD82 inhibited tumor growth in the xenograft model. The results demonstrated that the overexpression of KAI1/CD82 significantly inhibited the proliferation and invasion of human oral cancer cells, and inhibited tumor growth in the xenograft model. Therefore, KAI1/CD82 may be considered as a potential therapeutic target in oral cancer.

Involvement of intracellular transport in TREK-1c current run-up in 293T cells

ABSTRACTThe TREK-1 channel, the TWIK-1-related potassium (K+) channel, is a member of a family of 2-pore-domain K+ (K2P) channels, through which background or leak K+ currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335–360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.

Oncogenic miR-23a-5p is associated with cellular function in RCC.

In recent years, accumulating evidence has demonstrated that microRNAs (miRs, miRNAs) may serve an important role in the occurrence and development of tumors. miR‑23a‑5p has been confirmed as an oncogene in numerous diseases through gene chip analysis. However, as the most common type of renal tumor, the expression and function of miR‑23a‑5p in renal cell carcinoma (RCC) remains unclear. In the present study, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis, and Cell Counting Kit‑8 (CCK‑8), wound scratch, Transwell, MTT and flow cytometry assays were performed to investigate the role of miR‑23a‑5p in RCC. The expression of miR‑23a‑5p in RCC tissue samples was significantly higher compared with that in normal tissue samples (P<0.01). Furthermore, the expression of miR‑23a‑5p in RCC cell lines (786O, ACHN and Caki‑1) was significantly higher compared with that in the human embryo kidney 293T cell line, as determined using RT‑qPCR (P<0.001). In addition, the results revealed that the upregulation of miR‑23a‑5p promoted the proliferation, migration and invasion of RCC cells, and inhibited RCC cell apoptosis. The downregulation of miR‑23a‑5p resulted in the reversal of the results described above. Additionally, it was observed that the downregulation of miR‑23a‑5p significantly promoted ACHN and 786O cell viability (P<0.001). The results of the present study suggest that miR-23a-5p is an oncogene in the occurrence and development of RCC and may be a novel therapeutic target for RCC.

DNA damage response defect in Williams-Beuren syndrome

Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non-Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ-H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient-derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea-induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR-dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation.

A Fine-Tune Role of Mir-125a-5p on Foxn1 During Age-Associated Changes in the Thymus.

Decline of transcription factor FoxN1, which predominantly regulates thymic epithelial cell (TEC) differentiation and homeostasis lifelong, is demonstrated to be casually related to age-related thymic involution. Whereas, a global role of microRNAs (miRNAs) has also been demonstrated to control and maintain TEC-constituting thymic microenvironment and to be changed in expression profile in the aged thymus. Therefore, it is urgently necessary to build knowledge regarding whether and which miRNAs regulate FoxN1 gene in the aged thymus. We primarily compared changes in miRNA expression profile between young and aged murine TECs with Mus musculus miRBase-V20 arrays (containing 1892 unique probes), and clearly identified and validated that at least one miRNA, miR-125a-5p, was increased in aged thymus. Applying miR-125a-5p mimics was able to inhibit FoxN1 3′UTR luciferase activity in a 293T cell line and to suppress FoxN1 expression in murine TEC Z210 cells. Since a single miRNA can play a fine-tuning role to regulate expression of multiple genes and a single gene can be regulated by multiple miRNAs, our result adds a single miRNA, miR-125a-5p, into the panel of FoxN1-regulating miRNAs associated with thymic aging.

Synthesis and Cytotoxic Evaluation of Steroidal Copper (Cu (II)) Complexes

Using estrone and pregnenolone as starting materials, some steroidal copper complexes were synthesized by the condensation of steroidal ketones with thiosemicarbazide or diazanyl pyridine and then complexation of steroidal thiosemicarbazones or steroidal diazanyl pyridines with Cu (II). The complexes were characterized by IR, NMR, and HRMS. The synthesized compounds were screened for their cytotoxicity against HeLa, Bel-7404, and 293T cell lines in vitro. The results show that all steroidal copper (II) complexes display obvious antiproliferative activity against the tested cancer cells. The IC50 values of complexes 5 and 12 against Bel-7404 (human liver carcinoma) are 5.0 and 7.0 μM.

Exploration of Mutation and DNA Methylation of Polo-Like Kinase 1 (PLK1) in Colorectal Cancer

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays critical roles in cell cycling and DNA damage response. Overexpression of PLK1 is associated with the poorer prognosis of cancers, including colorectal cancer (CRC). Although the downstream pathways of the overexpression that lead to oncogenesis have been extensively studied, little is known about the factors that cause the overexpression of PLK1 in CRC. DNA methylation was reported to be affecting the expression of PLK1 in some cancers. The study aims to investigate the contribution of genetic mutation and DNA methylation of the PLK1 gene to the overexpression of PLK1 in CRC. The study involves data mining from Catalogue of Somatic Mutations in Cancer (COSMIC) and UniProtKB, Sanger sequencing on DNA from the cell lines HCT116, SW48, Colo320DM and T84 to analyse the possible mutation of PLK1. Other than that methylation status of the PLK1 promoter in CRC are also analysed by using mass spectrometry (MS) and pyrosequencing. Data from the COSMIC show the low incidences of PLK1 mutation for CRC (3.02%) with 46 mutations identified. One of the mutation p.R337Q (c.1010G > A) is located in the D-box which is an important motif for protein ubiquitination and eventually proteasomal degradation. Hence this mutation may potentially result in stabilisation of the PLK1 protein. Mutations are detected at the upstream silencer region, the promoter region and Exon1 in HCT116 but are not located at the protein binding or functioning site. Similarly, the same mutation at promoter region is detected in SW48. Differential trends of changes in methylation status of PLK1 in the IR treated CRC cell lines detected by MS reveal the possible association between the methylation and the radiosensitivity. Furthermore, pyrosequencing shows that PLK1 methylation status in tumour tissues with high expression of PLK1 is not significantly different from those with no PLK1 expression. In conclusion, mutation of PLK1 gene is infrequent in CRC and the expression of PLK1 is unlikely to be dependent on DNA methylation in the promoter region of PLK1 in the CRC.

LncRNA AFAP1-AS Functions as a Competing Endogenous RNA to Regulate RAP1B Expression by sponging miR-181a in the HSCR.

Background: Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in a broad spectrum of cellular processes including development and disease. Despite the known engagement of the AFAP1-AS in several human diseases, its biological function in Hirschsprung disease (HSCR) remains elusive.Methods: We used qRT-PCR to detect the relative expression of AFAP1-AS in 64 HSCR bowel tissues and matched normal intestinal tissues. The effects of AFAP1-AS on cell proliferation, migration, cell cycle, apoptosis and cytoskeletal organization were evaluated using CCK-8, transwell assay, flow cytometer analysis and immunofluorescence, in 293T and SH-SY5Y cell lines, respectively. Moreover, the competing endogenous RNA (ceRNA) activity of AFAP1-AS on miR-181a was investigated via luciferase reporter assay and immunoblot analysis.Results: Aberrant inhibition of AFAP1-AS was observed in HSCR tissues. Knockdown of AFAP1-AS in 293T and SH-SY5Y cells suppressed cell proliferation, migration, and induced the loss of cell stress filament integrity, possibly due to AFAP1-AS sequestering miR-181a in HSCR cells. Furthermore, AFAP1-AS could down-regulate RAP1B via its competing endogenous RNA (ceRNA) activity on miR-181a.Conclusions: These findings suggest that aberrant expression of lncRNA AFAP1-AS, a ceRNA of miR-181a, may involve in the onset and progression of HSCR by augmenting the miR-181a target gene, RAP1B.

Lactose-based glycoconjugates with variable spacers for design of liver-targeted liposomes

Asialoglycoprotein receptors are highly abundant on the hepatocyte surface and have specific binding sites for blood serum glycoproteins. Such discovery resulted in development of liver-targeted drug delivery systems because modification of the liposomal surface by carbohydrate derivatives results in an increase of endocytosis, which facilitates selective uptake of such systems by hepatocytes. In this study we have synthesized novel lactose derivatives containing a palmitic hydrophobic domain. They were used for modification of the liposome surface. Transfection activity of modified liposomes was analyzed on the HepG2 cell line (hepatocytes) and showed an increase in the transfection efficiency as compared to the non-modified liposomes. At the same time transfection activities of modified and non-modified liposomes were similar in the case of a non-hepatocyte cell line (293T). The novel lactose-based glycoconjugates may be a promising tool for developing efficient vectors for delivery of nucleic acids to hepatocytes.

HYKL-40 cancer biomarker electroanalysis in serum samples and model cell lysates: capacitive immunosensing compared with enzyme label immunosorbent assays (ELISA).

Human chitinase 3-like protein 1 (CHI3L1 or hYKL-40), a potential molecular marker for several cancers, was measured in clinical human serum samples and model cell lysates by indirect and competitive enzyme-linked immunosorbent assay (ELISA) and by capacitive immunosensing, so as to evaluate a recently introduced electrochemical method for routine use in cancer-monitoring studies. The clinical samples tested included serum from four healthy individuals, five breast cancer and four glioblastoma patients; cultures of the human monocytic cell line THP-1, known to secrete hYKL-40, and of the human embryonic kidney 293t cell line, which does not express hYKL-40, provided cell lysates and cell culture media for positive and negative bio- and electrochemical control trials. A good agreement was observed between the results of the three tested methods during hYKL-40 quantifications in human serum and cell lysates. Measurements of 'spiked' samples from healthy volunteers, cancer patients and hYKL-40-free 293t cell lysates revealed that capacitive immunosensing and the two types of ELISA all recovered the analyte with an efficiency close to 100%. On this basis, capacitive hYKL-40 immunosensor screening is a promising stand-alone or complementary analytical tool for the analysis of hYKL-40 in serum, and would be useful for the validation of standard ELISA data and also, because of the significantly lower hYKL-40 detection limit of the electroanalytical procedure, would permit assay of the marker and cancer observation at earlier stages than is currently possible using ELISA.

Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs)

Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

Increasing the Sensitivity of BCR-ABL Monitoring By Digital PCR By Reducing the Background Noise

BackgroundDroplet digital PCR (dPCR) is a potentially appealing technology for quantitative detection of specific mutations with simultaneous measurement of the reference gene and without the need for standard curves. This makes dPCR particularly suitable for minimal residual disease (MRD) diagnostics in chronic myeloid leukaemia. However, a limitation of the dPCR assays compared to standard quantitative PCR (qPCR) is the background (termed lower limit of blank, LoB) of 1 or 2 positive droplets for BCR-ABL (Franke et al, ASH 2015, Cross et al, Leukemia 2016). The resulting false positive rate (FPR) limits the sensitivity and the ability of the test to detect deep molecular remissions.AimsThe aim was to establish BCR-ABL (b2/a2 and/or b3/a2 transcript) dPCR assays with a sensitivity at least equivalent to that of qPCR, in which <5% of negative controls generate a single positive drop (FPR<5%, LoB=0).MethodsOne assay (Assay 1) was developed in Leipzig and another (Assay 2) by Bio-Rad laboratories. Sample preparation was identical for both assays up to the stage of reverse transcription (RT). Assay 1 used routine local RT procedure followed by a duplex BCR-ABL / ABL dPCR reaction modified from the M-BCR-ABL PCR published by Jennings et al. 2014. We tested ABL primer combinations from exons 4, 8, 10, and 11 generating a range of product lengths and optimised quenchers and PCR conditions to maximise the separation between positive and negative droplets. Assay 2 consisted of a reverse transcriptase reaction followed by a duplex PCR specific for ABL and both the b2/a2 and b3/a2 transcripts of BCR-ABL. Digital droplet PCR was performed in both cases using the Bio-Rad QX200 system.Background noise and cross hybridisation were assessed in non-template controls (NTC), BCR-ABL negative cell lines and healthy wild-type donor samples. In addition, assay 2 was tested on 40 CML patient samples with MRD levels ranging from BCR-ABL negative to 1% BCR-ABL/ABL as defined by standard qPCR.ResultsAssay 1 yielded a FPR of 4% (4/94) in NTC with 1 positive droplet for BCR-ABL and a FPR of 3% for ABL (n = 3/94), resulting in a specificity of >95% and a LOB of 0. The BCR-ABL FPR using wild type target (HEK 293T cell line and healthy donors) was 5% (5/92 and 2/37, respectively).Assay2: The specificity was >95% for both BCR-ABL and ABL in NTC and wild type samples. Extensive NTC analysis yielded no false positives for BCR-ABL PCR (n = 0/176; LoB = 0) and 1% false positives in ABL PCR (n = 2/176; 1-2 positive droplets, LoB = 0). The BCR-ABL negative cell line was confirmed negative for BCR-ABL PCR (n = 44), while the BCR-ABL assays of healthy donors were positive in 2% (n = 5/234) with 1 positive droplet per positive sample. The BCR-ABL copy numbers of the 40 CML samples were lower by dPCR than by RT-PCR (median 3/6) with similar ABL transcripts (median 55500/54949). This resulted in lower ratios by dPCR (mean 0.0313 vs. 0.0461 by qPCR). However, after conversion of the qPCR results with our lab specific conversion factor (CF) <1, the dPCR ratios were higher (mean 0.0313 vs. 0.0134). The MR classes were concordant in 60% of the samples, but 30% had more MRD while 10% had less MRD by dPCR than by qPCR, confirming our previous results using the EAC protocol (Franke, ASH 2015). More samples were MRD positive by dPCR than by qPCR (58% vs 50%).ConclusionsAn assay with a FPR of <5% and a LoB = 0 for the detection of BCR-ABL was developed. The BIO-RAD QX 200-Dx BCR-ABL Kit is fully functional and has a high specificity for BCR-ABL and ABL with a LoB of 0. The high specificity and low FPR/LoB of both assays enables sensitive and accurate monitoring of deep molecular remissions in CML. DisclosuresFranke:Novartis: Honoraria, Research Funding. Tzonev:Bio-Rad: Employment, Equity Ownership. Shelton:Bio-Rad: Employment. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau. Lange:Novartis: Research Funding.

English

Several investigations have demonstrated in vitro antibacterial activity of plant extracts. Kalanchoe pinnata is globally used in folk medicine for the treatment of various diseases, such as diarrhea, infections, tuberculosis and fever. Among the enteropathogenic bacteria, enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in the world. EAEC is also capable of forming biofilms, conformation that provides antibiotic resistance and a high degree of dispersion and reinfection, which may represent up to 80% of causes of human microbial infections. Thus, it becomes necessary to search for new antimicrobial agents with activity against biofilms. The aim of this study was to evaluate the cytotoxic, antibacterial and antibiofilm in EAEC from aqueous extracts of leaves and the flavonoids quercetin and rutin, occurring in K. pinnata. Leaf aqueous extracts were obtained and evaluated phytochemically. The cytotoxic activity was evaluated in human carcinoma cell lines HEp-2, Caco-2 and T84. The antibacterial activity was evaluated by the macrodilution method and the evaluation of antibiofilm activity was performed in Escherichia coli enteroaggregative (EAEC 042). Aqueous extracts of K pinnata do not show toxicity to Hep-2, but all other cell lines were sensitive to this extract. Instead, the flavonoids quercetin and rutin showed no cytotoxicity with any of the tested cell lines. Quercetin is capable of inhibiting bacterial growth of all tested strains. The aqueous leaf extract and quercetin were able to inhibit EAEC 042 biofilm formation above 50%. The results indicate the potential use of the species in treatment of bacterial infections. Key words: Antimicrobials, EAEC 042, biofilms, quercetin, Crassulaceae.