A method for transforming lymphocytes from very small blood volumes suitable for paediatric samples

Summary Permanent lymphoblastoid cell lines are important in the molecular analysis and characterization of human genetic disorders, when immortalized cells must be banked for future diagnostic or research purposes. However, routine methods for transformation using Epstein-Barr virus (EBV) require blood volumes that may be difficult to collect from clinically compromised neonates and small children. Here we report a modified transformation procedure utilizing blood samples of small volume (less than 1.0ml), which we have found to be particularly useful for the immortalization of lymphocytes destined for future molecular genetic studies.     

Human plasma accelerates immortalization of B lymphocytes by Epstein-Barr virus

Abstract.   Objective : Serum and plasma contain species-specific factors that modulate cell population growth and function, and that are required for proliferation of most cell cultures. Foetal calf serum (FCS) is the most common source of these growth factors. We studied the effect of human plasma (HP) on the immortalization process of B lymphocytes by Epstein–Barr virus (EBV) was studied. Materials and methods : The effect of HP as compared to FCS was done through assessment of cell proliferation. Results : It was found that HP (autologous and non-autologous plasma) is more effective than FCS in generating lymphoblastoid cell lines, regardless of EBV status of the donors: 65% of HP-supplemented cultures developed into lymphoblastoid cell lines by 7–14 culture days, as compared to 16% of cultures with FCS. In addition, 6% of HP-supplemented cultures did not achieve becoming lymphoblastoid cell lines by day 35 in comparison to 94% of cultures with FCS. The higher proliferative effect of HP was not altered by heat inactivation or filtration. HP maintained its proliferative activity at 4 °C over 8 months, thus indicating that HP contains a stable growth factor(s), which accelerates B-lymphocyte immortalization. Conclusion : The results support other studies that recommend the use of autologous plasma for tissue culture, mainly in the case of autologous transplantation. Furthermore, the use of HP allows preparation of lymphoblastoid cell lines from a small amount of peripheral blood in a shorter period of time and with a higher rate of success.     

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.     

Epstein-Barr virus-induced transformation of B cells for the diagnosis of genetic metabolic disorders--enumerative conditions for cryopreservation

Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.     

Sustained expression of the novel EBV-induced zinc finger gene, ZNFEB, is critical for the transition of B lymphocyte activation to oncogenic growth transformation.

EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C(2)H(2) (Krüppel-type) zinc finger gene, ZNF(EB), that is trans-activated early following EBV infection. In this study, we characterize ZNF(EB), including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNF(EB) maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies.     

Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro.

Injection of Epstein-Barr virus (EBV)-transformed human lymphoblastoid B cells into immunodeficient SCID mice results in the appearance of rapidly growing, fatal human B-cell tumors. To evaluate the role of EBV nuclear protein 2 (EBNA-2) in this process, we generated lymphoblastoid cell lines transformed by several EBV mutants which were identical except for deletions in the EBNA-2 gene (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991). These cell lines were injected intraperitoneally into SCID mice, and the interval until tumor detection was determined. Cell lines transformed with EBV type 1 (strain W91) or with EBV type 2 (strain P3HR-1) with an inserted type 1 EBNA-2 gene grew at the same rapid rate, indicating the potential importance of EBNA-2 for tumor formation in vivo. Cell lines derived from three different EBV mutants with deletions in the amino half of EBNA-2 produced tumors more slowly than cell lines transformed by wild-type W91 virus. In contrast, a cell line transformed with an EBV mutant with a deletion in the carboxy terminus of EBNA-2 grew more rapidly than cell lines transformed by wild-type virus. EBV mutants with deletions in the amino half of EBNA-2 had had reduced transforming activity in vitro, while the carboxy-terminal EBNA-2 mutant had had transforming activity greater than or equal to that of the wild type. These data indicate that EBNA-2 plays a critical role both for B-cell tumor growth in SCID mice and for B-lymphocyte transformation in vitro.     

Feasibility of using cryopreserved lymphoblastoid cells to diagnose some lysosomal storage diseases

Objectives: The Epstein–Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B‐lymphocytes. For this reason, EBV is used in conservation of human B‐lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1‐gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. Materials and methods: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes β‐galactosidase, β‐glucosidase, α‐iduronidase, α‐galactosidase and α‐glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. Results: We observed some significant alterations in enzymatic activity of non‐cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. Conclusions: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.     

Activation and infection of B cells by Epstein-Barr virus. Role of calcium mobilization and of protein kinase C translocation

Both the activation and the transformation of human B cells by EBV were inhibited by either the Ca2+ channel blocking agent verapamil or the combination of theophylline and dibutyryl cAMP: the day 4 and day 20 peaks of [3H]TdR incorporation were abolished; the EBNA marker was not expressed by day 10; lymphoblastoid cell lines did not arise. Short term incubation of B cells with EBV or verapamil showed that the effect of verapamil was reversible and took place early in the interaction between EBV and B cells. The effect of EBV on the early metabolic events of B cell response was thus examined in the presence and in the absence of the drugs. Compared to anti-mu stimulation, supernatant of the transforming B95-8 strain as well as that of the non-transforming P3HR1 strain induced a drug sensitive increase of the free cytosolic Ca2+ concentration. This increase was associated with a protein kinase C translocation from the cytosol to a membrane bound compartment. Moreover, B95-8 supernatant induced phosphatidyl inositol metabolism by human B cells but at least four times less than that induced by anti-mu antibody. These metabolic events induced by EBV were significantly inhibited by anti-CD21 antibodies whereas anti-mu induced metabolic events were not. The infection of EBV negative Ramos cell line was prevented by verapamil or by theophylline + dibutyryl cAMP. Verapamil did not modify the density of EBV receptors but negatively interfered with the penetration of the virus into B cells. Thus B cell activation through the EBV receptor and virus penetration share a common metabolic pathway which is also used for transduction of the signal delivered through the membrane Ig.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

An Epstein-Barr virus isolated from a lymphoblastoid cell line has a 16-kilobase-pair deletion which includes gp350 and the Epstein-Barr virus nuclear antigen 3A

Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.     

Signal transduction through the B cell antigen receptor is normal in ataxia-telangiectasia B lymphocytes.

The rare human genetic disorder ataxia-telangiectasia (A-T) has multiple consequences including a variable degree of immunodeficiency. Khanna and co-workers (Khanna, K. K., Yan, J., Watters, D., Hobson, K., Beamish, H., Spring, K., Shiloh, Y., Gatti, R. A., and Lavin, M. F. (1997) J. Biol. Chem. 272, 9489-9495) evaluated signaling in Epstein-Barr virus (EBV) immortalized A-T lymphoblastoid cell lines (LCLs), derived from the B cells of A-T patients. They showed that A-T lymphoblastoid cells lack signaling through the B cell antigen receptor and concluded that the fault in A-T encompasses intracellular signaling in B cells. However, it is established that EBV latent membrane protein 2A (LMP2A) blocks signaling in EBV-bearing cells by interaction with cellular tyrosine kinases. To test whether the reported fault in A-T B cells was not inherent in A-T but the result of influence of wild-type EBV, we derived A-T LCLs with wild-type or LMP2A-deleted EBV and studied signaling in these cells in response to cross-linking the B cell antigen receptor. We report that intracellular calcium mobilization and tyrosine phosphorylation in LMP2A-depleted LCLs derived from A-T patients is indistinguishable from that in LMP2A-depleted LCLs derived from normal controls. Further, signaling is blocked similarly in A-T and normal lymphoblastoid cells bearing wild-type EBV. In conclusion there is no evidence of any defect in B cell receptor signal transduction in A-T B cells.     

Human blymphocytes immortalization by epstein-barr virus in the presence of cyclosporin a

Summary A simple method was devised for the establishment of continuous lymphoblastoid, cell lines (LCL). Unfractionated mononuclear cells collected from healthy donors were infected in vitro by Epstein-Barr Virus (EBV) (strain B95-8) under specific, conditions: an immunosuppressive drug, Cyclosporin-A (CS-A, Sandoz), associated with the use of a feeder layer (MRC-5) led to 100% efficiency of LCL establishment. A bank of 400 LCL was set up for completion of genetic studies. Regression and kinetics of virus-induced transformation were monitored and related to donors' EBV immune status. Mean time of LCL establishment and probability of regression among seropositive donors were not linked to any given value titer of antibodies against Viral Capsid Antigen (VCA) or against Epstein-Barr Nuclear Antigen (EBNA). However, when the anti-VCA:anti-EBNA ratio was considered, this parameter, seemed to be linked to the kinetics of transformation but not to the probability of regression. Once LCL are established, large quantities of human cells can be produced. The complete cellular DNA is available so that any part of it can be scrutinized. Moreover, some of the phenotypic characteristics of these B cells be used for a wide range of investigations. The work was supported by Fondation de la Recherche Medicale (France) for Professor J. Dausset (Paris) and by the International Agency for Research on Cancer (Lyon).     

Epstein-Barr virus preferentially induces proliferation of primed B cells

EBV can induce human B cells to proliferate, differentiate, and undergo transformation into continuously growing lymphoblastoid cell lines. The EBV responsiveness appears to be confined to a very limited subpopulation of B cells, the nature of which is still unclear. In these studies, we sorted tonsillar B cells on the basis of their expression of the early surface activation Ag, Bac-1, and compared their proliferative responses to EBV. Bac-1+ cells responded to EBV with a relatively high level of DNA synthesis, whereas the Bac-1- cells did not. Both large and small Bac-1+ cells were responsive to EBV and the responsiveness was unrelated to the level of Bac-1 immunofluorescence intensity. Bac-1+ cells were relatively enriched for surface IgM and IgD expression. When the Bac-1- population was enriched for IgM+ cells, the proliferative response was still significantly lower than that of the Bac-1+ population. B cells acquire the ability to bind IgM relatively late after activation, and this feature did not distinguish the EBV-responsive B cells. The results suggest B cells become responsive to EBV after an early activation signal.     

Assessment of the effect of TLR7/8, TLR9 agonists and CD40 ligand on the transformation efficiency of Epstein-Barr virus in human B lymphocytes by limiting dilution assay

Infection of human B cells with Epstein-Barr virus (EBV) induces polyclonal activation in almost all infected cells, but a small proportion of infected cells are transformed to immortalized lymphoblastoid cell lines. Since B cells are activated also by CD40 ligand (CD40L) and Toll-like receptor (TLR) agonists via a similar signaling pathway, it is likely that costimulation through these molecules could result in synergistic enhancement of the transformation efficiency of EBV. In this study, the stimulatory effect of TLR7/8 (R848), TLR9 (CpG) agonists and/or CD40L on transformation efficiency of EBV in normal human B cells was assessed using the limiting dilution assay. Costimulation of peripheral blood mononuclear cells (PBMCs) with CpG and R848, but not CD40L, increased significantly the frequency of EBV transformed B cells (p < 0.001). Neither synergistic nor additive effects were observed between TLR agonists and CD40L and also TLR7/8 and TLR9 agonists. Costimulation with R848, CpG and CD40L enhanced the proliferative response of B cells infected with EBV. This effect was more evident when enriched B cells were employed, compared to PBMCs. The promoting effect of TLR agonists stimulation, implies that EBV may take advantage of the genes induced by the TLR stimulation pathway for viral latency and oncogenesis.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Phosphonoacetic acid: inhibition of transformation of human cord blood lymphocytes by Epstein-Barr virus.

Phosphonoacetic acid disodium salt (PAA) inhibited the transformation of human cord blood lymphocytes by Epstein-Barr virus (EBV) at concentrations of 50-100 microgram/ml. At these concentrations, PAA had no effect on the multiplication of EBV transformed human lymphoblastoid cells or on the survival of human cord blood lymphocytes. The transformation of human cord blood lymphocytes by the B95-8 strain of EBV was measured by 3H-thymidine uptake, 5 days or more after infection. The degree of inhibition of transformation was correlated with the relation between the input of EBV and the concentration of PAA in the experiment. PAA inhibited the transformation even when added 24 h after EBV infection, but had no effect when added 48 h after EBV infection. The inhibitory effect of PAA could be overcome by its removal and normal 3H-thymidine uptake was restored even after 6 days of inhibition. The specificity of the inhibitory effect on EBV induced transformation of human cord blood lymphocytes is discussed.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.