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International audience; More than 80 mutations in the skeletal muscle ryanodine receptor gene have been found to be associated with autosomal dominant forms of malignant hyperthermia and central core disease, and with recessive forms of multi-minicore disease. Studies on the functional effects of pathogenic dominant mutations have shown that they mostly affect intracellular Ca2+ homoeostasis, either by rendering the channel hypersensitive to activation (malignant hyperthermia) or by altering the amount of Ca2+ released subsequent to physiological or pharmacological activation (central core disease). In the present paper, we show, for the first time, data on the functional effect of two recently identified recessive ryanodine receptor 1 amino acid substitutions, P3527S and V4849I, as well as that of R999H, another substitution that was identified in two siblings that were affected by multi-minicore disease. We studied the intracellular Ca2+ homoeostasis of EBV (Epstein-Barr virus)-transformed lymphoblastoid cells from the affected patients, their healthy relatives and control individuals. Our results show that the P3527S substitution in the homozygous state affected the amount of Ca2+ released after pharmacological activation with 4-chloro-m-cresol and caffeine, but did not affect the size of the thapsigargin-sensitive Ca2+ stores. The other substitutions had no effect on either the size of the intracellular Ca2+ stores, or on the amount of Ca2+ released after ryanodine receptor activation; however, both the P3527S and V4849I substitutions had a small but significant effect on the resting Ca2+ concentration.

Most studies looking at the reversal of drug resistance in hematological malignancies have focused on the blockade of P-glycoprotein (Pgp) pump activity, as one of the more important proteins involved in cell resistance to the chemotherapeutic agents used. In model experiments using the IM-9 lymphoblastoid cell line cultured with increasing doses of vincristine (Vcr) in the presence or absence of the Pgp blocker cyclosporin A (CsA) we established two Vcr resistant sublines. By measuring Pgp expression in target cells, and the activity and sensitivity to cytostatics we believe that our results suggest the presence of other mechanisms involved in cell resistance to xenobiotics and that these mechanisms should be considered in the development of clinical trials designed to overcome drug resistance.

Epstein–Barr virus lacking glycoprotein gp85 cannot infect B-cells and epithelial cells. The gp85 belongs to the molecular complex required for virus invasion of B-lymphocyte or epithelial cells. Moreover, there is evidence that gp85 is necessary for virus attachment to epithelial cells. Thirty-six peptides from the entire gp85-sequence were tested in epithelial and lymphoblastoid cell line binding assays to identify gp85-regions involved in virus–cell interaction. Five of these peptides presented high binding activity to Raji, Ramos, P3HR-1, and HeLa cells, but not to erythrocytes; Raji-cell affinity constants were between 80 and 140 nM. Of these five peptides, 11435 (181TYKRVTEKGDEHVLSLVFGK200), 11436 (201TKDLPDLRGPFSYPSLTSAQ220), and 11438 (241YFVPNLKDMFSRAVTMTAAS260) bound to a 65 kDa protein on Raji-cell surface. These peptides and antibodies induced by them (recognising live EBV-infected cells) inhibited Epstein–Barr virus interaction with cord blood lymphocytes. It is thus probable that gp85-regions defined by peptides 11435, 11436, and 11438 are involved in EBV invasion of B-lymphocytes.

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 microM, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 microM. In the medium used for the CBMN assay, quercetin (at 100 microM) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 microM), quercetin-3-O-sulfate (at 10 microM), and isorhamnetin (at 5 microM) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.

Using a human lymphoblastoid cell line WTK-1, we applied multicolor fluorescence in situ hybridization (mFISH) technique to analyze mitomycin C (MMC)-induced chromatid exchanges, focusing especially on the triradial chromosomes. It was found that the triradial chromosomes were formed with a specific rearrangement, "recipient and donor" relationship. The exchange sites of the recipient chromosomes were on single chromatid breaks and distributed randomly throughout the interstitial, pericentromeric, and terminal regions. In counterpart, donor chromosomes exchanged on isochromatid breaks of their telomeric and/or subtelomeric regions with the single chromatid breaks of recipient chromosomes. More than 80% of the scored triradial chromosomes were formed with such rearrangements, and few acentric chromosome fragments derived from the donor chromosomes could be detected in the metaphases observed. We therefore suggest that biological mechanisms of breakages between the recipient and donor chromosomes are different: the former due to direct DNA-damage by MMC, but the latter due to indirect DNA-damage depending on telomeric specific structure/function.

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific “nested” primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Telomerase has been studied extensively in human and murine tumors, but little is known about the role of telomerase in the tumor biology of other vertebrate species such as the chicken. We studied the telomerase activity of the lymphoblastoid cell line derived from lymphomas induced by Marek's disease virus (MDCC-MSB1) compared with another avian cell line (PA5) and peripheral blood lymphocytes (PBL) using the telomeric repeat amplification protocol (TRAP) Assay. Telomerase activity in MDCC-MSB1 was 4.5 times greater than in the PA5 cell line and normal avian lymphocytes. These results demonstrate for the first time that telomerase is more intense in one transformed cell line than in normal cells, suggesting a potential role for telomerase in carcinogenesis induced by an avian virus.

Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4+/CD25− primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis. Cancer Prev Res; 5(4); 685–96. ©2012 AACR.

Summary An oligonucleotide containing multiple AP-1 binding sites was introduced into the regulatory sequence in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV). Chloramphenicol acetyltransferase assay revealed that basal promoter activity of the mutated LTR was higher than that of the wild-type LTR in Crandell feline kidney (CRFK) cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the clone was transfected into CRFK cells. The virus production of the mutant in the cells was as high as that of the wild-type when determined by the reverse transcriptase activity assay. The growth of the mutant virus obtained from the transfected CRFK cells was examined in feline T lymphoblastoid cell lines (MYA-1 and FeL-039 cells) and primary feline peripheral blood mononuclear cells (fPBMCs). The growth was delayed when compared with that of the wild-type virus in all the cells used. Upon examination by polymerase chain reaction, the length of the LTR of the mutant virus was shortened in both MYA-1 cells and fPBMCs. Sequence analysis revealed that the insertion was completely deleted 39 days after infection in the MYA-1 cells.

Background A detailed analysis of whole genomes can be now achieved with next generation sequencing. Epstein Barr Virus (EBV) transformation is a widely used strategy in clinical research to obtain an unlimited source of a subject’s DNA. Although the mechanism of transformation and immortalization by EBV is relatively well known at the transcriptional and proteomic level, the genetic consequences of EBV transformation are less well understood. A detailed analysis of the genetic alterations introduced by EBV transformation is highly relevant, as it will inform on the usefulness and limitations of this approach. Results We used whole genome sequencing to assess the genomic signature of a low-passage lymphoblastoid cell line (LCL). Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL. A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the normal genomic DNA. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels). Conclusions Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias.

The purpose of this study was to elucidate if the cellular DNA repair capacity, genotypes of DNA repair genes and the occurrence of breast cancer can be associated with each other on the same set of probands in a single study. Rarely characterized features of the DNA repair capacity measures were studied as a prerequisite for their use in the definition as an intermediate phenotype of cancer. The study population comprised different cohorts with sporadic or familial breast cancer cases and controls. DNA repair assays were performed in parallel on peripheral blood samples of the probands: the baseline and the radiation induced micronucleus (MN) assay, the baseline and the induced sister chromatid exchange (SCE) assay and the mitotic delay (MD) assay. 23 missense variants in 15 genes were genotyped. An increased induced MN frequency and a decreased MD index were associated with the occurrence of breast cancer. The minor allele of the variant Slx4 S1271F (rs3810813) was enriched in breast cancer cases compared to controls (OR ~ 1.6) and displayed an increase with younger age of the cases. In-silico analysis revealed a probable functional impact on the Slx4 protein. The same variant was also associated with an increase of the induced MN frequency. The minor allele of the variant rs13044759 in the NINL gene was associated with an increased MD index. The measured DNA repair end points are largely independent and can be used to characterize different aspects of the cellular DNA repair capacity. Lymphoblastoid cell lines are not applicable as surrogates for peripheral blood lymphocytes as source of study material. Heritability estimates showed that the MN assays and the MD assay predominantly reflect the influence of genetic factors. Thus, the concordance between the results of the three types of association studies concerning the induced MN and the MD assay supports the view that the cellular DNA repair capacity can be of use as an intermediate phenotype of breast cancer.

The analysis of free amino acids in mammalian cell culture media can give valuable information on the metabolism of particular commercially valuable cell lines. Analysis of spent culture media indicates potential limiting nutrients. However, analysis over the whole culture period enables a kinetic approach to allow understanding of amino acid uptake and potential strategies to improve media design. This communication details the use of a less commonly used HPLC protocol, indicating various advantages and disadvantages. Further, the batch kinetics of amino acid metabolism of two cell lines are discussed.

Intraepithelial leukocytes (IELs) form an important component of the intestinal immune system. Several methods of preparing chicken IELs were compared for cell yield, cell populations, and functional activity against a natural killer (NK)-susceptible lymphoblastoid cell line (LSCC-RP9). In addition, an attempt was made to immortalize IELs by infection with reticuloendotheliosis virus (REV) for use in cell-trafficking studies. Intestinal fragments were incubated in 5 mM DL-dithiothreitol at 41 degrees C for 15 min followed by three 45 min incubations in 0.1 mM EDTA. Gentle shaking of the intestinal fragments every 5 min caused considerably less damage to the epithelial cell layer than slow stirring with a magnetic bar. The latter method yielded more cells, but these were not functionally active in the NK-cell assay. The more gentle method resulted in a lower cell yield, but these cells were able to lyse LSCC-RP9; the percentage of lymphocytes present was lower but these contained a higher proportion of large granular lymphocytes. Infection of IELs with REV did not immortalize these cells.

In this report we describe a type D virus isolated from a human neuroblastoma cell line (Paju). The viral RNA was isolated, partially molecularly cloned and sequenced. Our clones were shown to be identical to a human D-type retrovirus previously isolated from a human lymphoblastoid cell line. However, we obtained no evidence for the virus in earlier passage of the Paju cell line and therefore we must consider this isolate a laboratory contamination. contamination.

The rapid increase of the applications for Lab- on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale devel- opment, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), exten- sively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70–75%, but only after a prolonged exposure of 3–6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper ? 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the mate- rials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.

Objectives: Epstein-Barr virus (EBV) transformation has been described as a routine method to establish human B lymphoblastoid cell lines. Each established lymphoblastoid cell line represents one unique genetic information carrier and can produce unlimited quantities of DNA materials available for downstream applications and research. Undoubtedly, it is of great value to human clinical and experimental genetic studies. However, the current process of EBV transformation requires much manpower in the routine renewal of medium, which is time-consuming. This situation can become a serious problem especially when establishing a human B lymphoblastoid cell bank. A modified and cost-effective protocol for EBV transformation should be considered. Materials and methods: In the present study, process in EBV transformation was modified to fit the requirements of robot handling. Results: 1 mL of whole blood was demonstrated to be sufficient to perform EBV transformation. Additionally, EBV transformation can performed in 96-deep-well plates that are directly and widely used with automatic work platforms. Conclusions: Based on these facts, a process of EBV transformation can be modified to fit the requirements of robot handling.

Lymphoblastoid cell lines (LCL) are being actively and extensively used to examine the expression of specific genes and (genome-wide expression profiles, including allele specific expression assays. However, it has recently been shown that approximately 10% of human genes exhibit random patterns of monoallelic expression within single clones of LCLs. Consequently allelic imbalance studies could be significantly compromised if bulk populations of donor cells are clonal, or near clonal. Here, using X chromosome inactivation as a readout, we confirm and quantify widespread near monoclonality in two independent sets of cell lines. Consequently, we recommend where possible the use of bulk, non cell line, ex vivo cells for allele specific expression assays. © 2008 Plagnol et al.

Mice are not able to respond in making specific antibodies against a variety of human antigens. One of these antigens is the erythrocyte D-antigen of the Rh-system. Until now, it has been possible only to make murine monoclonals against structures of the Rh-proteins. These antibodies were not suitable for routine use in blood grouping. As an alternative, the human immune system is able to produce alloantibodies against Rh-D with high specificity. As a result, with the development of reproducible methods for the generation of human monoclonals, we have started to establish protocols for the production of antibodies against the Rh-D antigen. In addition, we selected a heteromyeloma line, MD33, which is a non-secretor line with high fusion rate giving rise to hybridomas which are high producers. Here, we compare different methods for their efficiency in producing human monoclonals against the Rh-D antigen. We tested the direct fusion of human PBL with either mouse myeloma or human lymphoblastoid cell line, immortalization by EBV-transformation, EBV-transformation followed by fusion with mouse myeloma line, and EBV-transformation followed by fusion with heteromyeloma line. We could demonstrate that most success can be achieved by fusions of EBV-transformed PBL with mouse myeloma line P3X63-Ag8.653 and heteromyeloma line MD33 which resulted in several stable and antigen-specific lines per fusion.

In order to remove the EAI "blocking" activity, EA inhibition positive sera from patients with recurrent abortions were absorbed after alloimmunization with paternal lymphocytes by the lymphoblastoid cell line (LCL) of the husband. To avoid non-specific binding via the Fc-receptor, the cells were first fixed with 0.05% glutaraldehyde. Absorption was performed for 3 h at 4 degrees C. For subsequent elution, the cells were incubated with 0.1 M glycine-HCl, pH 2.3, for 30 min at 4 degrees C. After each step, EAI assay was carried out to determine the "blocking" activity in the supernatants. Furthermore, 10% SDS-PAGE and immunoblotting with goat antihuman IgG of the whole serum and the supernatants were performed. The results obtained give evidence that the EAI "blocking" activity can be absorbed by and eluted from the LCL of the immunizing husband, and is due to an IgG antibody directed against an antigen present on the LCL. Further absorption experiments with trophoblast cells will show whether this antigen is also displayed by the trophoblast.

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis. The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.

A B-lymphoblastoid cell line (Ts-B) was established from lymph nodes of an apparently healthy cynomolgus monkey. The cells were demonstrated to contain Epstein-Barr virus-related antigens. Moreover, herpesvirus particles were found in the cells by electron microscopy. The cell-free culture medium transformed lymphocytes of cynomolgus rhesus monkeys, and of Japanese monkeys.

At present, CTL are being used only in patients with advanced relapsed malignancies. As their safety and efficacy becomes better established we may expect their introduction earlier in the course of therapy where they may reduce the short- and long-term toxicities of standard radio- and chemotherapies. Further understanding of the ways in which immune evasion strategies can be counteracted in Hodgkin’s disease may also be applied to the many other human tumors that are potentially immunogenic and use similar immune evasion strategies.

Enhancing effect of vitamin A acetate on antibody productivity was examined using human-human hybridoma cell line AE6, BD9 and C5. The effect was maintained for about two weeks by treatment of A E6 hybridomas with 1 μg/ml of vitamin A acetate for a day, and thereafter recovered by its another treatment. The enhancement of antibody production was influenced by cell densities of AE6 hybridomas: two- to four-fold enhancement was observed in cell densities under 106 cells/ml, but hardly observed in those over 108 cells/ml. Therefore, vitamin A acetate might be suitable for middle scale production of human monoclonal antibody by hybridomas sustained at relatively low cell densities. In addition, enhancing effect of vitamin A acetate was also observed in BD9 hybridomas as well as AE6 hybridomas, but not in C5 hybridomas. These observations indicated that the enhancing effect might be limited by hybridoma cell lines.

The analysis of a lymphoblastoid cell line (5106), derived from a rare individual of normal intelligence with an unmethylated full mutation of the FMR1 gene, allowed us to reconstruct the chain of molecular events leading to the FMR1 inactivation and to fragile X syndrome. We found that lack of DNA methylation of the entire promoter region, including the expanded CGG repeat, correlates with methylation of lysine 4 residue on the N-tail of histone H3 (H3-K4), as in normal controls. Normal levels of FMR1 mRNA were detected by real-time fluorescent RT-PCR (0.8-1.4 times compared with a control sample), but mRNA translation was less efficient (-40%), as judged by polysome profiling, resulting in reduced levels of FMRP protein (similar to30% of a normal control). These results underline once more that CGG repeat amplification per se does not prevent FMR1 transcription and FMRP production in the absence of DNA methylation. Surprisingly, we found by chromatin immunoprecipitation that cell line 5106 has deacetylated histones H3 and H4 as well as methylated lysine 9 on histone H3 (H3-K9), like fragile X cell lines, in both the promoter and exon 1. This indicates that these two epigenetic marks (i.e. histone deacetylation and H3-K9 methylation) can be established in the absence of DNA methylation and do not interfere with active gene transcription, contrary to expectation. Our results also suggest that the molecular pathways regulating DNA and H3-K4 methylation are independent from those regulating histone acetylation and H3-K9 methylation. ispartof: Human Molecular Genetics vol:14 issue:2 pages:267-277 ispartof: location:England status: published